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  • 99
    Beckman Coulter gallios flow cytometer
    Percentage of CD45+ leukocyte sub‐sets in peripheral blood from non‐pregnant and pregnant women. Peripheral blood was collected into Cyto‐Chex tubes and stained with different leukocyte markers (CD14, CD15, CD3, CD4, CD8, CD19, CD56 and CD16) to identify ( A ) granulocytes, ( B ) monocytes, ( C ) T cells, ( D ) B cells and ( E ) NK cells. Flow cytometry data were acquired by <t>Gallios</t> cytometer followed with analysis by Kaluza 1.3 software. The distribution of different leukocyte sub‐sets (% from total CD45+ leukocytes) was calculated. Data are presented as mean ± SD. Significant difference between non‐pregnant and pregnant women is indicated by *, P
    Gallios Flow Cytometer, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 99/100, based on 6581 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gallios flow cytometer/product/Beckman Coulter
    Average 99 stars, based on 6581 article reviews
    Price from $9.99 to $1999.99
    gallios flow cytometer - by Bioz Stars, 2019-05
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    99
    Millipore imaging flow cytometry
    Identification and characterization of monocyte subsets in whole blood. Using flow <t>cytometry,</t> monocyte subsets were identified directly in whole blood based on their expression patterns of CD14 and CD16 as described in the Methods section. (a) Gating for the identification of monocyte subsets was based on the successive exclusion of platelets, red blood cells (RBC), granulocytes, and lymphocytes on bivariate scatter plots. The remaining population was discriminated on a CD14 vs . CD16 scatter plot to discriminate three monocyte subsets, classical (CM), intermediate (IM), and non-classical (NCM) monocytes (n = 5). (b) Monocyte subsets were further characterized based on their expression of chemokine receptors CCR2, CX 3 CR1, and CCR5 (n = 3). MFI, mean fluorescence intensity. (c) Phenotypic characterization of monocyte subsets based on their expression of CD14, CD16, CCR2, CX 3 CR1, and CCR5.
    Imaging Flow Cytometry, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/imaging flow cytometry/product/Millipore
    Average 99 stars, based on 148 article reviews
    Price from $9.99 to $1999.99
    imaging flow cytometry - by Bioz Stars, 2019-05
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    99
    Becton Dickinson facs canto ii flow cytometer
    Induction of GIL-specific T cell responses by different concentrations of GIL and whole-inactivated influenza virus (WIV). HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with indicated amounts of GIL peptide and WIV. (A) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine <t>IFN-γ.</t> The percentages for IFN-γ + CD8 + T cells acquired by <t>FACS</t> are plotted. (B) Splenocytes of immunized mice were re-stimulated with GIL peptide. The number of CD8 + IFN-γ-secreting cells was subsequently determined with ELISpot. (A,B) Data are shown as mean ± SD of three mice per group, each dot is a single replicate and data are from a single experiment. * p
    Facs Canto Ii Flow Cytometer, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 5530 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/facs canto ii flow cytometer/product/Becton Dickinson
    Average 99 stars, based on 5530 article reviews
    Price from $9.99 to $1999.99
    facs canto ii flow cytometer - by Bioz Stars, 2019-05
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    99
    Becton Dickinson facs calibur flow cytometer
    (A-D) CD68 stained cells. Comparisons of CD68-stained cells in different cell culture media [(A and C) DMEM F12 and (B and D) RPMI] between TCPS control plates (C and D) and Repellent plates (A and B) analysed in the gated MF. Stained cells are shown as dark grey line against the background of the light grey shaded isotype control (all suspension cells, donor 14). Measurement was carried out with the BD <t>FACS</t> <t>Calibur</t> Flow Cytometer and data were analysed with the FlowJo 10 program. CD, cluster of differentiation; DMEM F12, Dulbecco's modified Eagle's medium nutrient mixture F-12; RPMI, Roswell Park Memorial Institute Medium; TCPS, tissue culture plates; MF, monocyte fraction; BD, Becton Dickinson; FACS, fluorescence-activated cell sorting.
    Facs Calibur Flow Cytometer, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 17777 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/facs calibur flow cytometer/product/Becton Dickinson
    Average 99 stars, based on 17777 article reviews
    Price from $9.99 to $1999.99
    facs calibur flow cytometer - by Bioz Stars, 2019-05
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    99
    Becton Dickinson facscalibur flow cytometer
    Dasatinib blocks antigen-induced TCR downregulation and tetramer internalization from the cell surface. A. Mel13 CTL were pre-treated with PBS ± 50 nM dasatinib and exposed to C1R-A2 B cells previously pulsed with 10 -6 M ELAGIGILTV peptide or medium alone for 4 h at 37 °C. Cells were subsequently stained with anti-TCR-FITC (clone BMA 031; Serotec) and anti-CD8-APC (clone RPA-T8; BD Pharmingen) mAbs for 30 min on ice, washed twice and resuspended in PBS. Data were acquired on a <t>FACSCalibur</t> flow cytometer (BD) and analyzed using FlowJo software. B. 10 5 ILA1 CTL were pre-treated with PBS (i ii) or PBS + 50 nM dasatinib (iii iv) for 30 min at 37 °C, then stained with 20μg/ml HLA A2/ILAKFLHWL-Alexa488 tetramer for 15 min at 37 °C. Microscopy was performed as described in the Materials and methods.
    Facscalibur Flow Cytometer, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 64261 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/facscalibur flow cytometer/product/Becton Dickinson
    Average 99 stars, based on 64261 article reviews
    Price from $9.99 to $1999.99
    facscalibur flow cytometer - by Bioz Stars, 2019-05
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    99
    Becton Dickinson facs aria flow cytometer
    Effect of different TLR2 complex agonists on the proliferation of <t>GFP</t> + Treg. <t>FACS-sorted</t> GFP + Treg (5×10 4 ) were cultured w ith plate-bound anti-CD3 (5µg/ml) and IL-2 for 3 days in the presence or absence of: A , TLR2/TLR2 agonist, LTA-SA;
    Facs Aria Flow Cytometer, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 1565 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/facs aria flow cytometer/product/Becton Dickinson
    Average 99 stars, based on 1565 article reviews
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    facs aria flow cytometer - by Bioz Stars, 2019-05
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    87
    Incyte guava easycyte ht flow cytometer
    LPS and Con A-stimulated bovine PBMC. PBMC were separated from venous blood of one cow and immediately submitted to LPS and Con A stimulation assays, respectively. After three days in culture, non-adherent PBMC (lymphocytes) were fixed overnight at -20°C in 70% ethanol and stained with PI in the presence of RNAse for 30 min at room temperature. PBMC were analyzed in a Guava <t>EasyCyte</t> HT flow cytometer using software “Cell Cycle” (Merck Millipore). The percentages of S phase cells of control, LPS-stimulated and Con A-stimulated PBMC are shown as mean ± 1 standard deviation of three test replicates. The percentage of S phase cells was significantly higher after Con A stimulation (P
    Guava Easycyte Ht Flow Cytometer, supplied by Incyte, used in various techniques. Bioz Stars score: 87/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/guava easycyte ht flow cytometer/product/Incyte
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    guava easycyte ht flow cytometer - by Bioz Stars, 2019-05
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    Image Search Results


    Percentage of CD45+ leukocyte sub‐sets in peripheral blood from non‐pregnant and pregnant women. Peripheral blood was collected into Cyto‐Chex tubes and stained with different leukocyte markers (CD14, CD15, CD3, CD4, CD8, CD19, CD56 and CD16) to identify ( A ) granulocytes, ( B ) monocytes, ( C ) T cells, ( D ) B cells and ( E ) NK cells. Flow cytometry data were acquired by Gallios cytometer followed with analysis by Kaluza 1.3 software. The distribution of different leukocyte sub‐sets (% from total CD45+ leukocytes) was calculated. Data are presented as mean ± SD. Significant difference between non‐pregnant and pregnant women is indicated by *, P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Immunophenotyping and activation status of maternal peripheral blood leukocytes during pregnancy and labour, both term and preterm

    doi: 10.1111/jcmm.13160

    Figure Lengend Snippet: Percentage of CD45+ leukocyte sub‐sets in peripheral blood from non‐pregnant and pregnant women. Peripheral blood was collected into Cyto‐Chex tubes and stained with different leukocyte markers (CD14, CD15, CD3, CD4, CD8, CD19, CD56 and CD16) to identify ( A ) granulocytes, ( B ) monocytes, ( C ) T cells, ( D ) B cells and ( E ) NK cells. Flow cytometry data were acquired by Gallios cytometer followed with analysis by Kaluza 1.3 software. The distribution of different leukocyte sub‐sets (% from total CD45+ leukocytes) was calculated. Data are presented as mean ± SD. Significant difference between non‐pregnant and pregnant women is indicated by *, P

    Article Snippet: Freshly prepared FACS Lyse solution (450 μl; BD Bioscience, San Jose, CA, USA) was added to each tube and incubated for 15 min. prior to data acquisition by FACSAria (BD Biosciences) or by Gallios flow cytometer (Beckman Coulter, Brea, CA, USA).

    Techniques: Staining, Flow Cytometry, Cytometry, Software

    The activation status of circulating leukocytes among preterm not‐in‐labour (PTNIL), PTL (PTL), term not‐in‐labour (TNIL) and term labour (TL) pregnancy. Peripheral blood was collected in Cyto‐Chex tubes and stained with leukocyte surface markers (CD14, CD15, CD3, CD4, CD8 and CD19) and activation markers (CD11b, CD44, CD55, CD181 and CD192) to assess the activation status of ( A ) CD15+ granulocytes, ( B ) CD14+ monocytes, ( C ) CD3+ T cells, ( D ) CD4+CD3+ T cells, ( E ) CD8 + CD3+ T cells and ( F ) CD19+ B cells. The percentages of positive sub‐sets (top panel) and their signal intensity mean fluorescent intensity (MFI) (bottom panel) are shown as box plots. Flow cytometry data were acquired by Gallios cytometer followed with analysis. *, P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Immunophenotyping and activation status of maternal peripheral blood leukocytes during pregnancy and labour, both term and preterm

    doi: 10.1111/jcmm.13160

    Figure Lengend Snippet: The activation status of circulating leukocytes among preterm not‐in‐labour (PTNIL), PTL (PTL), term not‐in‐labour (TNIL) and term labour (TL) pregnancy. Peripheral blood was collected in Cyto‐Chex tubes and stained with leukocyte surface markers (CD14, CD15, CD3, CD4, CD8 and CD19) and activation markers (CD11b, CD44, CD55, CD181 and CD192) to assess the activation status of ( A ) CD15+ granulocytes, ( B ) CD14+ monocytes, ( C ) CD3+ T cells, ( D ) CD4+CD3+ T cells, ( E ) CD8 + CD3+ T cells and ( F ) CD19+ B cells. The percentages of positive sub‐sets (top panel) and their signal intensity mean fluorescent intensity (MFI) (bottom panel) are shown as box plots. Flow cytometry data were acquired by Gallios cytometer followed with analysis. *, P

    Article Snippet: Freshly prepared FACS Lyse solution (450 μl; BD Bioscience, San Jose, CA, USA) was added to each tube and incubated for 15 min. prior to data acquisition by FACSAria (BD Biosciences) or by Gallios flow cytometer (Beckman Coulter, Brea, CA, USA).

    Techniques: Activation Assay, Staining, Flow Cytometry, Cytometry

    The activation status of circulating CD45+ leukocytes in peripheral blood of non‐pregnant and pregnant women. Peripheral blood was collected in Cyto‐Chex tubes and stained with leukocyte surface and activation markers (CD11b, CD44, CD55, CD181 and CD192) to assess the immunological characteristics of ( A ) CD15+ granulocytes ( B ) CD14+ monocytes, ( C ) CD3+ T cells, ( D ) CD4 + CD3+ T cells, ( E ) CD8+CD3+ T and ( F ) CD19+ B cells. The percentages of positive sub‐sets (top panel) and their signal intensity mean fluorescent intensity (MFI) (bottom panel) are shown as box plots. Flow cytometry data were acquired by Gallios cytometer followed with analysis by Kaluza 1.3 software. Significant difference between non‐pregnant and pregnant women is indicated, when applicable, by *, P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Immunophenotyping and activation status of maternal peripheral blood leukocytes during pregnancy and labour, both term and preterm

    doi: 10.1111/jcmm.13160

    Figure Lengend Snippet: The activation status of circulating CD45+ leukocytes in peripheral blood of non‐pregnant and pregnant women. Peripheral blood was collected in Cyto‐Chex tubes and stained with leukocyte surface and activation markers (CD11b, CD44, CD55, CD181 and CD192) to assess the immunological characteristics of ( A ) CD15+ granulocytes ( B ) CD14+ monocytes, ( C ) CD3+ T cells, ( D ) CD4 + CD3+ T cells, ( E ) CD8+CD3+ T and ( F ) CD19+ B cells. The percentages of positive sub‐sets (top panel) and their signal intensity mean fluorescent intensity (MFI) (bottom panel) are shown as box plots. Flow cytometry data were acquired by Gallios cytometer followed with analysis by Kaluza 1.3 software. Significant difference between non‐pregnant and pregnant women is indicated, when applicable, by *, P

    Article Snippet: Freshly prepared FACS Lyse solution (450 μl; BD Bioscience, San Jose, CA, USA) was added to each tube and incubated for 15 min. prior to data acquisition by FACSAria (BD Biosciences) or by Gallios flow cytometer (Beckman Coulter, Brea, CA, USA).

    Techniques: Activation Assay, Staining, Flow Cytometry, Cytometry, Software

    The activation status of circulating CD45+ leukocytes in maternal blood of pregnant (1st/2nd/3rd trimester and post‐dates) and term labouring (TL) women. Peripheral blood was collected in Cyto‐Chex tubes and stained with leukocyte surface markers (CD14, CD15, CD3, CD4, CD8 and CD19) and activation markers (CD11b, CD44, CD55, CD181 and CD192) to assess activation status of ( A ) CD15+ granulocytes, ( B ) CD14+ monocytes, ( C ) CD3+ T cells, ( D ) CD4+CD3+ T cells, ( E ) CD8+CD3+ T cells and ( F ) CD19+ B cells. The percentages of positive sub‐sets (top panel) and their signal intensity mean fluorescent intensity (MFI) (bottom panel) are shown as box plots. Flow cytometry data were acquired by Gallios cytometer and are presented as mean ± SD. Significant difference in the activation markers between early and late gestation as well as between pregnant and labouring women is indicated by *, P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Immunophenotyping and activation status of maternal peripheral blood leukocytes during pregnancy and labour, both term and preterm

    doi: 10.1111/jcmm.13160

    Figure Lengend Snippet: The activation status of circulating CD45+ leukocytes in maternal blood of pregnant (1st/2nd/3rd trimester and post‐dates) and term labouring (TL) women. Peripheral blood was collected in Cyto‐Chex tubes and stained with leukocyte surface markers (CD14, CD15, CD3, CD4, CD8 and CD19) and activation markers (CD11b, CD44, CD55, CD181 and CD192) to assess activation status of ( A ) CD15+ granulocytes, ( B ) CD14+ monocytes, ( C ) CD3+ T cells, ( D ) CD4+CD3+ T cells, ( E ) CD8+CD3+ T cells and ( F ) CD19+ B cells. The percentages of positive sub‐sets (top panel) and their signal intensity mean fluorescent intensity (MFI) (bottom panel) are shown as box plots. Flow cytometry data were acquired by Gallios cytometer and are presented as mean ± SD. Significant difference in the activation markers between early and late gestation as well as between pregnant and labouring women is indicated by *, P

    Article Snippet: Freshly prepared FACS Lyse solution (450 μl; BD Bioscience, San Jose, CA, USA) was added to each tube and incubated for 15 min. prior to data acquisition by FACSAria (BD Biosciences) or by Gallios flow cytometer (Beckman Coulter, Brea, CA, USA).

    Techniques: Activation Assay, Staining, Flow Cytometry, Cytometry

    Identification and characterization of monocyte subsets in whole blood. Using flow cytometry, monocyte subsets were identified directly in whole blood based on their expression patterns of CD14 and CD16 as described in the Methods section. (a) Gating for the identification of monocyte subsets was based on the successive exclusion of platelets, red blood cells (RBC), granulocytes, and lymphocytes on bivariate scatter plots. The remaining population was discriminated on a CD14 vs . CD16 scatter plot to discriminate three monocyte subsets, classical (CM), intermediate (IM), and non-classical (NCM) monocytes (n = 5). (b) Monocyte subsets were further characterized based on their expression of chemokine receptors CCR2, CX 3 CR1, and CCR5 (n = 3). MFI, mean fluorescence intensity. (c) Phenotypic characterization of monocyte subsets based on their expression of CD14, CD16, CCR2, CX 3 CR1, and CCR5.

    Journal: Scientific Reports

    Article Title: Differential Interaction of Platelet-Derived Extracellular Vesicles with Leukocyte Subsets in Human Whole Blood

    doi: 10.1038/s41598-018-25047-x

    Figure Lengend Snippet: Identification and characterization of monocyte subsets in whole blood. Using flow cytometry, monocyte subsets were identified directly in whole blood based on their expression patterns of CD14 and CD16 as described in the Methods section. (a) Gating for the identification of monocyte subsets was based on the successive exclusion of platelets, red blood cells (RBC), granulocytes, and lymphocytes on bivariate scatter plots. The remaining population was discriminated on a CD14 vs . CD16 scatter plot to discriminate three monocyte subsets, classical (CM), intermediate (IM), and non-classical (NCM) monocytes (n = 5). (b) Monocyte subsets were further characterized based on their expression of chemokine receptors CCR2, CX 3 CR1, and CCR5 (n = 3). MFI, mean fluorescence intensity. (c) Phenotypic characterization of monocyte subsets based on their expression of CD14, CD16, CCR2, CX 3 CR1, and CCR5.

    Article Snippet: In addition to their flow cytometric characterization in plasma, EVs were analyzed directly in heparinized whole blood and their interaction with blood cells was assessed by Imaging Flow Cytometry (ImageStreamx MkII cytometer, INSPIRE v6.1, Amnis, EMD Millipore, Seattle, WA) .

    Techniques: Flow Cytometry, Cytometry, Expressing, Fluorescence

    Release of extracellular vesicles (EVs) during storage of whole blood vs . platelet-free plasma (PFP). (a) Scheme summarizing the experimental set-up. Freshly drawn whole blood anticoagulated with heparin or citrate was either centrifuged immediately to obtain fresh PFP, or stored (3 h, 37 °C) prior to the generation of PFP. For comparison, PFP was stored (3 h, 37 °C) prior to analysis. EVs were characterized by flow cytometry using CD41 as marker for platelet origin, CD235a as marker for red blood cell origin, and lactadherin as marker for phosphatidylserine exposure as described in the Methods section. (b) Quantification of EVs in fresh PFP, stored PFP, and in PFP from stored whole blood revealed a significant release of platelet-derived EVs during the storage of heparinized whole blood (n = 3, p ≤ 0.05; paired t -test). Representative flow cytometry scatter plots are given in Supplementary Fig. S2 .

    Journal: Scientific Reports

    Article Title: Differential Interaction of Platelet-Derived Extracellular Vesicles with Leukocyte Subsets in Human Whole Blood

    doi: 10.1038/s41598-018-25047-x

    Figure Lengend Snippet: Release of extracellular vesicles (EVs) during storage of whole blood vs . platelet-free plasma (PFP). (a) Scheme summarizing the experimental set-up. Freshly drawn whole blood anticoagulated with heparin or citrate was either centrifuged immediately to obtain fresh PFP, or stored (3 h, 37 °C) prior to the generation of PFP. For comparison, PFP was stored (3 h, 37 °C) prior to analysis. EVs were characterized by flow cytometry using CD41 as marker for platelet origin, CD235a as marker for red blood cell origin, and lactadherin as marker for phosphatidylserine exposure as described in the Methods section. (b) Quantification of EVs in fresh PFP, stored PFP, and in PFP from stored whole blood revealed a significant release of platelet-derived EVs during the storage of heparinized whole blood (n = 3, p ≤ 0.05; paired t -test). Representative flow cytometry scatter plots are given in Supplementary Fig. S2 .

    Article Snippet: In addition to their flow cytometric characterization in plasma, EVs were analyzed directly in heparinized whole blood and their interaction with blood cells was assessed by Imaging Flow Cytometry (ImageStreamx MkII cytometer, INSPIRE v6.1, Amnis, EMD Millipore, Seattle, WA) .

    Techniques: Flow Cytometry, Cytometry, Marker, Derivative Assay

    Differential interaction of EVs with monocyte subsets. Monocyte subsets were characterized using flow cytometry as shown in Fig. 4 . Classical (CM), intermediate (IM), and non-classical (NCM) monocytes associated with platelet-derived EVs were identified as CD41 + lactadherin + cells (a) in freshly drawn whole blood and (b) after storage of whole blood for 3 h, as shown on representative scatter plots; (c) percentages of classical, intermediate, and non-classical monocytes associated with EVs (average of five independent experiments). A total of 2,000 events were recorded for each monocyte subset. At both time points, intermediate monocytes showed significantly higher association with EVs as compared to non-classical monocytes (n = 5; p ≤ 0.05; Friedman test).

    Journal: Scientific Reports

    Article Title: Differential Interaction of Platelet-Derived Extracellular Vesicles with Leukocyte Subsets in Human Whole Blood

    doi: 10.1038/s41598-018-25047-x

    Figure Lengend Snippet: Differential interaction of EVs with monocyte subsets. Monocyte subsets were characterized using flow cytometry as shown in Fig. 4 . Classical (CM), intermediate (IM), and non-classical (NCM) monocytes associated with platelet-derived EVs were identified as CD41 + lactadherin + cells (a) in freshly drawn whole blood and (b) after storage of whole blood for 3 h, as shown on representative scatter plots; (c) percentages of classical, intermediate, and non-classical monocytes associated with EVs (average of five independent experiments). A total of 2,000 events were recorded for each monocyte subset. At both time points, intermediate monocytes showed significantly higher association with EVs as compared to non-classical monocytes (n = 5; p ≤ 0.05; Friedman test).

    Article Snippet: In addition to their flow cytometric characterization in plasma, EVs were analyzed directly in heparinized whole blood and their interaction with blood cells was assessed by Imaging Flow Cytometry (ImageStreamx MkII cytometer, INSPIRE v6.1, Amnis, EMD Millipore, Seattle, WA) .

    Techniques: Flow Cytometry, Cytometry, Derivative Assay

    Visualization of EV-immune cell interactions using imaging flow cytometry. Imaging flow cytometry was performed after staining of blood cells using CD45-PB and CD14-PE as monocyte markers, CD16/56-PC5 as granulocyte and NK cell marker, and CD3-ECD as T cell marker. EVs were traced with lactadherin (LA)-FITC and CD41-PC7 for platelet EVs as well as with lactadherin-FITC and CD235a-APC-AF750 for red blood cell EVs as described in the Methods section. (a) Cells in focus were selected (gradient RMS_BF > 50); (b) leukocytes were identified within the focused events based on their expression patterns of CD45 and CD14; (c) a scatter plot of aspect ratio and area in bright field (BF) was used to define single cells within double positive focused events (aspect ratio > 0.8); (d) monocytes, granulocytes, and lymphocytes were identified based on their expression of CD45 and CD14 as described in the Methods section; (e) T cells were discriminated based on their expression of CD3 vs . CD45; (f) monocytes and granulocytes interacted with platelet EVs, whereas no association of EVs with T cells was detected; (g) EVs were identified as CD41 + lactadherin + events for platelet origin or CD235a + lactadherin + events for red blood cell origin. Statistical significance was assessed using the Wilcoxon matched-pairs signed rank test for monocytes and T cells (not normally distributed data) or paired t -test for granulocytes (normally distributed data). n = 3, p ≤ 0.05. The whole panel including monocytes, granulocytes, NK cells, T cells, and B cells is shown in Supplementary Fig. S2 . Scale bar, 7 µm.

    Journal: Scientific Reports

    Article Title: Differential Interaction of Platelet-Derived Extracellular Vesicles with Leukocyte Subsets in Human Whole Blood

    doi: 10.1038/s41598-018-25047-x

    Figure Lengend Snippet: Visualization of EV-immune cell interactions using imaging flow cytometry. Imaging flow cytometry was performed after staining of blood cells using CD45-PB and CD14-PE as monocyte markers, CD16/56-PC5 as granulocyte and NK cell marker, and CD3-ECD as T cell marker. EVs were traced with lactadherin (LA)-FITC and CD41-PC7 for platelet EVs as well as with lactadherin-FITC and CD235a-APC-AF750 for red blood cell EVs as described in the Methods section. (a) Cells in focus were selected (gradient RMS_BF > 50); (b) leukocytes were identified within the focused events based on their expression patterns of CD45 and CD14; (c) a scatter plot of aspect ratio and area in bright field (BF) was used to define single cells within double positive focused events (aspect ratio > 0.8); (d) monocytes, granulocytes, and lymphocytes were identified based on their expression of CD45 and CD14 as described in the Methods section; (e) T cells were discriminated based on their expression of CD3 vs . CD45; (f) monocytes and granulocytes interacted with platelet EVs, whereas no association of EVs with T cells was detected; (g) EVs were identified as CD41 + lactadherin + events for platelet origin or CD235a + lactadherin + events for red blood cell origin. Statistical significance was assessed using the Wilcoxon matched-pairs signed rank test for monocytes and T cells (not normally distributed data) or paired t -test for granulocytes (normally distributed data). n = 3, p ≤ 0.05. The whole panel including monocytes, granulocytes, NK cells, T cells, and B cells is shown in Supplementary Fig. S2 . Scale bar, 7 µm.

    Article Snippet: In addition to their flow cytometric characterization in plasma, EVs were analyzed directly in heparinized whole blood and their interaction with blood cells was assessed by Imaging Flow Cytometry (ImageStreamx MkII cytometer, INSPIRE v6.1, Amnis, EMD Millipore, Seattle, WA) .

    Techniques: Imaging, Flow Cytometry, Cytometry, Staining, Marker, Expressing

    Induction of GIL-specific T cell responses by different concentrations of GIL and whole-inactivated influenza virus (WIV). HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with indicated amounts of GIL peptide and WIV. (A) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (B) Splenocytes of immunized mice were re-stimulated with GIL peptide. The number of CD8 + IFN-γ-secreting cells was subsequently determined with ELISpot. (A,B) Data are shown as mean ± SD of three mice per group, each dot is a single replicate and data are from a single experiment. * p

    Journal: Frontiers in Immunology

    Article Title: Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes

    doi: 10.3389/fimmu.2018.00525

    Figure Lengend Snippet: Induction of GIL-specific T cell responses by different concentrations of GIL and whole-inactivated influenza virus (WIV). HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with indicated amounts of GIL peptide and WIV. (A) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (B) Splenocytes of immunized mice were re-stimulated with GIL peptide. The number of CD8 + IFN-γ-secreting cells was subsequently determined with ELISpot. (A,B) Data are shown as mean ± SD of three mice per group, each dot is a single replicate and data are from a single experiment. * p

    Article Snippet: Finally, cells were stained intracellularly with anti-mouse IFN-γ-APC (BD Biosciences), and IFN-γ+ CD8+ T cells were quantified on a FACS Canto II flow cytometer (BD Biosciences).

    Techniques: Transgenic Assay, Mouse Assay, Staining, FACS, Enzyme-linked Immunospot

    Comparison of CD8 + T cell responses induced by 1 nmol GIL peptide adjuvanted with either 50 µg CpG or 50 µg whole-inactivated influenza virus (WIV). HLA-A2.1 transgenic mice were injected twice, 3 weeks apart with PBS (negative control), WIV or GIL peptide with indicated adjuvant. Shown are the responses 2 weeks after the final immunization. (A) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. Data are shown as mean ± SD of three mice per group, each circle is a single replicate and data are from a single experiment representative of two individual experiments. ** p

    Journal: Frontiers in Immunology

    Article Title: Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes

    doi: 10.3389/fimmu.2018.00525

    Figure Lengend Snippet: Comparison of CD8 + T cell responses induced by 1 nmol GIL peptide adjuvanted with either 50 µg CpG or 50 µg whole-inactivated influenza virus (WIV). HLA-A2.1 transgenic mice were injected twice, 3 weeks apart with PBS (negative control), WIV or GIL peptide with indicated adjuvant. Shown are the responses 2 weeks after the final immunization. (A) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. Data are shown as mean ± SD of three mice per group, each circle is a single replicate and data are from a single experiment representative of two individual experiments. ** p

    Article Snippet: Finally, cells were stained intracellularly with anti-mouse IFN-γ-APC (BD Biosciences), and IFN-γ+ CD8+ T cells were quantified on a FACS Canto II flow cytometer (BD Biosciences).

    Techniques: Transgenic Assay, Mouse Assay, Injection, Negative Control, Staining, FACS

    Influence of co-localization on the immunogenicity of GIL (100 nmol) and whole-inactivated influenza virus (WIV, 25 µg). (A) HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with GIL peptide and WIV either combined in one single flank (mixed) or separate in opposite flanks (separate). (B) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (C) Splenocytes were re-stimulated with GIL peptide. The number of IFN-γ-secreting cells was subsequently determined with ELISpot. (A,B) Data are shown as mean ± SD of six mice per group, each dot is a single replicate and data are from a single experiment. ** p

    Journal: Frontiers in Immunology

    Article Title: Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes

    doi: 10.3389/fimmu.2018.00525

    Figure Lengend Snippet: Influence of co-localization on the immunogenicity of GIL (100 nmol) and whole-inactivated influenza virus (WIV, 25 µg). (A) HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with GIL peptide and WIV either combined in one single flank (mixed) or separate in opposite flanks (separate). (B) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (C) Splenocytes were re-stimulated with GIL peptide. The number of IFN-γ-secreting cells was subsequently determined with ELISpot. (A,B) Data are shown as mean ± SD of six mice per group, each dot is a single replicate and data are from a single experiment. ** p

    Article Snippet: Finally, cells were stained intracellularly with anti-mouse IFN-γ-APC (BD Biosciences), and IFN-γ+ CD8+ T cells were quantified on a FACS Canto II flow cytometer (BD Biosciences).

    Techniques: Transgenic Assay, Mouse Assay, Staining, FACS, Enzyme-linked Immunospot

    Effect of membrane fusion activity on whole-inactivated influenza virus (WIV) adjuvant activity. (A) WIV was fusion-inactivated by brief exposure to a buffer with pH 4.5 at 37°C. Hemolysis was performed by mixing either WIV (“active” WIV) or fusion-inactivated WIV (“inactive” WIV) with human blood erythrocytes at various pHs. Fusion-mediated hemoglobin release was subsequently determined by spectrophotometric analysis. Data are shown as mean ± SD of three individual experiments. (B) HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with GIL peptide (100 µg) and either fusion-active or fusion-inactive WIV (25 µg). Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (C) Splenocytes were re-stimulated with GIL peptide. The number of IFN-γ-secreting cells was subsequently determined with ELISpot. (B,C) Data are shown as mean ± SD of six mice per group, each dot is a single replicate and data are from a single experiment. n.s. = not significant (one-way ANOVA).

    Journal: Frontiers in Immunology

    Article Title: Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes

    doi: 10.3389/fimmu.2018.00525

    Figure Lengend Snippet: Effect of membrane fusion activity on whole-inactivated influenza virus (WIV) adjuvant activity. (A) WIV was fusion-inactivated by brief exposure to a buffer with pH 4.5 at 37°C. Hemolysis was performed by mixing either WIV (“active” WIV) or fusion-inactivated WIV (“inactive” WIV) with human blood erythrocytes at various pHs. Fusion-mediated hemoglobin release was subsequently determined by spectrophotometric analysis. Data are shown as mean ± SD of three individual experiments. (B) HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with GIL peptide (100 µg) and either fusion-active or fusion-inactive WIV (25 µg). Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (C) Splenocytes were re-stimulated with GIL peptide. The number of IFN-γ-secreting cells was subsequently determined with ELISpot. (B,C) Data are shown as mean ± SD of six mice per group, each dot is a single replicate and data are from a single experiment. n.s. = not significant (one-way ANOVA).

    Article Snippet: Finally, cells were stained intracellularly with anti-mouse IFN-γ-APC (BD Biosciences), and IFN-γ+ CD8+ T cells were quantified on a FACS Canto II flow cytometer (BD Biosciences).

    Techniques: Activity Assay, Transgenic Assay, Mouse Assay, Staining, FACS, Enzyme-linked Immunospot

    (A-D) CD68 stained cells. Comparisons of CD68-stained cells in different cell culture media [(A and C) DMEM F12 and (B and D) RPMI] between TCPS control plates (C and D) and Repellent plates (A and B) analysed in the gated MF. Stained cells are shown as dark grey line against the background of the light grey shaded isotype control (all suspension cells, donor 14). Measurement was carried out with the BD FACS Calibur Flow Cytometer and data were analysed with the FlowJo 10 program. CD, cluster of differentiation; DMEM F12, Dulbecco's modified Eagle's medium nutrient mixture F-12; RPMI, Roswell Park Memorial Institute Medium; TCPS, tissue culture plates; MF, monocyte fraction; BD, Becton Dickinson; FACS, fluorescence-activated cell sorting.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Comparison of different cell culture plates for the enrichment of non-adherent human mononuclear cells

    doi: 10.3892/etm.2019.7204

    Figure Lengend Snippet: (A-D) CD68 stained cells. Comparisons of CD68-stained cells in different cell culture media [(A and C) DMEM F12 and (B and D) RPMI] between TCPS control plates (C and D) and Repellent plates (A and B) analysed in the gated MF. Stained cells are shown as dark grey line against the background of the light grey shaded isotype control (all suspension cells, donor 14). Measurement was carried out with the BD FACS Calibur Flow Cytometer and data were analysed with the FlowJo 10 program. CD, cluster of differentiation; DMEM F12, Dulbecco's modified Eagle's medium nutrient mixture F-12; RPMI, Roswell Park Memorial Institute Medium; TCPS, tissue culture plates; MF, monocyte fraction; BD, Becton Dickinson; FACS, fluorescence-activated cell sorting.

    Article Snippet: Flow cytometry was performed immediately on a FACS calibur flow cytometer (Becton; BD Biosciences, Franklin Lakes, NJ, USA).

    Techniques: Staining, Cell Culture, FACS, Flow Cytometry, Cytometry, Modification, Fluorescence

    Percentage of CD68 + cells per total cells as determined by flow cytometry after cultivation in different cell culture plates with (A) DMEM F12 or (B) RPMI. Cells (1×10 5 ) were stained with 0.1 µg of CD68 antibodies as well as the isotype control IgG2 and measured with the BD FACS Calibur Flow Cytometer. Data were analysed with the FlowJo 10 program and values are depicted as box plots with minimum, 25th percentile, median, 75th percentile and maximum of CD68 positive cells per total cells for the individual donors and experiments (n≥4). Statistical analysis was performed using Kruskal-Wallis test with Dunn's multiple comparisons test as post hoc test. CD, cluster of differentiation; DMEM F12, Dulbecco's modified Eagle's medium nutrient mixture F-12; RPMI, Roswell Park Memorial Institute Medium; IgG2, immunoglobulin G2; BD, Becton Dickinson; FACS, fluorescence-activated cell sorting; TCPS, tissue culture plates.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Comparison of different cell culture plates for the enrichment of non-adherent human mononuclear cells

    doi: 10.3892/etm.2019.7204

    Figure Lengend Snippet: Percentage of CD68 + cells per total cells as determined by flow cytometry after cultivation in different cell culture plates with (A) DMEM F12 or (B) RPMI. Cells (1×10 5 ) were stained with 0.1 µg of CD68 antibodies as well as the isotype control IgG2 and measured with the BD FACS Calibur Flow Cytometer. Data were analysed with the FlowJo 10 program and values are depicted as box plots with minimum, 25th percentile, median, 75th percentile and maximum of CD68 positive cells per total cells for the individual donors and experiments (n≥4). Statistical analysis was performed using Kruskal-Wallis test with Dunn's multiple comparisons test as post hoc test. CD, cluster of differentiation; DMEM F12, Dulbecco's modified Eagle's medium nutrient mixture F-12; RPMI, Roswell Park Memorial Institute Medium; IgG2, immunoglobulin G2; BD, Becton Dickinson; FACS, fluorescence-activated cell sorting; TCPS, tissue culture plates.

    Article Snippet: Flow cytometry was performed immediately on a FACS calibur flow cytometer (Becton; BD Biosciences, Franklin Lakes, NJ, USA).

    Techniques: Flow Cytometry, Cytometry, Cell Culture, Staining, FACS, Modification, Fluorescence

    Percentage of CD14 + cells per total cells as determined by flow cytometry after cultivation in different cell culture plates with (A) DMEM F12 or (B) RPMI. Cells (1×10 5 ) were stained with 0.1 µg of CD14 antibodies as well as the isotype control IgG1 and measured with the BD FACS Calibur Flow Cytometer. Data were analysed with the FlowJo 10 program and values are depicted as box plots with minimum, 25th percentile, median, 75th percentile and maximum of CD14 positive cells per total cells for the individual donors and experiments (n≥4). Statistical analysis was performed using Kruskal-Wallis test with Dunn's multiple comparisons test as post hoc test. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Comparison of different cell culture plates for the enrichment of non-adherent human mononuclear cells

    doi: 10.3892/etm.2019.7204

    Figure Lengend Snippet: Percentage of CD14 + cells per total cells as determined by flow cytometry after cultivation in different cell culture plates with (A) DMEM F12 or (B) RPMI. Cells (1×10 5 ) were stained with 0.1 µg of CD14 antibodies as well as the isotype control IgG1 and measured with the BD FACS Calibur Flow Cytometer. Data were analysed with the FlowJo 10 program and values are depicted as box plots with minimum, 25th percentile, median, 75th percentile and maximum of CD14 positive cells per total cells for the individual donors and experiments (n≥4). Statistical analysis was performed using Kruskal-Wallis test with Dunn's multiple comparisons test as post hoc test. *P

    Article Snippet: Flow cytometry was performed immediately on a FACS calibur flow cytometer (Becton; BD Biosciences, Franklin Lakes, NJ, USA).

    Techniques: Flow Cytometry, Cytometry, Cell Culture, Staining, FACS

    Percentage of cells in the monocyte fraction as gated with FSC and SSC after cultivation in different cell culture plates with (A) DMEM F12 or (B) RPMI. Measurement was carried out with the BD FACS Calibur Flow Cytometer and data were analysed with the FlowJo 10 program. Values are depicted as box plots with minimum, 25th percentile, median, 75th percentile and maximum of the gated cell number of the individual donors and experiments (n≥4). Statistical analysis was performed using Two-way ANOVA followed by Tukey's multiple comparisons as post test. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Comparison of different cell culture plates for the enrichment of non-adherent human mononuclear cells

    doi: 10.3892/etm.2019.7204

    Figure Lengend Snippet: Percentage of cells in the monocyte fraction as gated with FSC and SSC after cultivation in different cell culture plates with (A) DMEM F12 or (B) RPMI. Measurement was carried out with the BD FACS Calibur Flow Cytometer and data were analysed with the FlowJo 10 program. Values are depicted as box plots with minimum, 25th percentile, median, 75th percentile and maximum of the gated cell number of the individual donors and experiments (n≥4). Statistical analysis was performed using Two-way ANOVA followed by Tukey's multiple comparisons as post test. *P

    Article Snippet: Flow cytometry was performed immediately on a FACS calibur flow cytometer (Becton; BD Biosciences, Franklin Lakes, NJ, USA).

    Techniques: Cell Culture, FACS, Flow Cytometry, Cytometry

    (A-E) Representative depiction of CD14 staining. (A) FSC and SSC gated MF overlaid with CD14 staining shown as blue dots (donor 14). Examples of comparison of CD14 stained cells in different cell culture media DMEM F12 (B and D) and RPMI (C and E) between TCPS control plates (B and C) and Repellent plates (D and E) analysed in the gated MF. Stained cells are shown as dark grey line against the background of the light grey shaded isotype control (all suspension cells, donor 14). Measurement was carried out with the BD FACS Calibur Flow Cytometer and data were analysed with the FlowJo 10 program. CD, cluster of differentiation; FSC, forward scatter; SSC, sideward scatter; MF, monocyte fraction; DMEM F12, Dulbecco's modified Eagle's medium nutrient mixture F-12; RPMI, Roswell Park Memorial Institute Medium; TCPS, tissue culture plates; BD, Becton Dickinson; FACS, fluorescence-activated cell sorting.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Comparison of different cell culture plates for the enrichment of non-adherent human mononuclear cells

    doi: 10.3892/etm.2019.7204

    Figure Lengend Snippet: (A-E) Representative depiction of CD14 staining. (A) FSC and SSC gated MF overlaid with CD14 staining shown as blue dots (donor 14). Examples of comparison of CD14 stained cells in different cell culture media DMEM F12 (B and D) and RPMI (C and E) between TCPS control plates (B and C) and Repellent plates (D and E) analysed in the gated MF. Stained cells are shown as dark grey line against the background of the light grey shaded isotype control (all suspension cells, donor 14). Measurement was carried out with the BD FACS Calibur Flow Cytometer and data were analysed with the FlowJo 10 program. CD, cluster of differentiation; FSC, forward scatter; SSC, sideward scatter; MF, monocyte fraction; DMEM F12, Dulbecco's modified Eagle's medium nutrient mixture F-12; RPMI, Roswell Park Memorial Institute Medium; TCPS, tissue culture plates; BD, Becton Dickinson; FACS, fluorescence-activated cell sorting.

    Article Snippet: Flow cytometry was performed immediately on a FACS calibur flow cytometer (Becton; BD Biosciences, Franklin Lakes, NJ, USA).

    Techniques: Staining, Cell Culture, FACS, Flow Cytometry, Cytometry, Modification, Fluorescence

    FasL expression on MVs purified from melanoma cell supernatant. (A) Western blot analysis. Analysis of MVs isolated from melanoma cell supernatant by serial ultracentrifugations, as performed by Western blot and staining with G247 mAb. An ∼35-kD band can be observed in MVs from melanoma lines (lanes 4, 6, and 7), resembling the one observed in MVs from PHA-activated Jurkat cells (lane 2). Only MV from PHA-activated, and not from resting Jurkat cells (lane 3) showed positivity for FasL, suggesting that this molecule unlikely derived from FBS used for cell culture. (B) Purity controls of microvesicle preparations. Controls performed to check purity of microvesicle preparations from melanoma cell supernatant revealed the expression of the melanosomal marker gp100, and no significant contamination with Golgi, ER, or mitochondria. (C) Cytofluorimetric analysis of FasL expression on purified MVs. MVs from 501mel line were stained with anti-FasL NOK-1, anti-gp100, anti–LAMP-2, and anti-CD63 mAbs, followed by incubation with FITC goat anti–mouse IgG (dark areas); an irrelevant isotype-matched Ab plus the FITC-conjugated goat anti–mouse IgG were used as negative control (clear areas). Fluorescence was analyzed by FACSCalibur™ and CELLQuest™ software. MVs were detected as granules with a size range of 100–600 nm, relative to standard beads of 6 μm size.

    Journal: The Journal of Experimental Medicine

    Article Title: Induction of Lymphocyte Apoptosis by Tumor Cell Secretion of FasL-bearing Microvesicles

    doi: 10.1084/jem.20011624

    Figure Lengend Snippet: FasL expression on MVs purified from melanoma cell supernatant. (A) Western blot analysis. Analysis of MVs isolated from melanoma cell supernatant by serial ultracentrifugations, as performed by Western blot and staining with G247 mAb. An ∼35-kD band can be observed in MVs from melanoma lines (lanes 4, 6, and 7), resembling the one observed in MVs from PHA-activated Jurkat cells (lane 2). Only MV from PHA-activated, and not from resting Jurkat cells (lane 3) showed positivity for FasL, suggesting that this molecule unlikely derived from FBS used for cell culture. (B) Purity controls of microvesicle preparations. Controls performed to check purity of microvesicle preparations from melanoma cell supernatant revealed the expression of the melanosomal marker gp100, and no significant contamination with Golgi, ER, or mitochondria. (C) Cytofluorimetric analysis of FasL expression on purified MVs. MVs from 501mel line were stained with anti-FasL NOK-1, anti-gp100, anti–LAMP-2, and anti-CD63 mAbs, followed by incubation with FITC goat anti–mouse IgG (dark areas); an irrelevant isotype-matched Ab plus the FITC-conjugated goat anti–mouse IgG were used as negative control (clear areas). Fluorescence was analyzed by FACSCalibur™ and CELLQuest™ software. MVs were detected as granules with a size range of 100–600 nm, relative to standard beads of 6 μm size.

    Article Snippet: The red fluorescence of individual nuclei was measured with a FACSCalibur™ flow cytometer (Becton Dickinson).

    Techniques: Expressing, Purification, Western Blot, Isolation, Staining, Derivative Assay, Cell Culture, Marker, Incubation, Negative Control, Fluorescence, Software

    FasL expression in melanosomes purified from melanoma cells. (A) Western blot analysis of purified melanosome preparations. Western blot analysis was performed using proteins derived from melanosomal preparations purified from melanoma lines by sucrose gradient. Staining with G247 mAb revealed the presence in melanosome preparations of the 40–33 kD band pattern (lane 3–5). (B) Purity controls of melanosome preparations. Controls performed to check purity of melanosome preparations revealed the expression of the melanosomal marker gp100, and no significant contamination with Golgi, mitochondria, or plasma-membrane markers. The ER marker Bip/GRP78 was detectable in melanosome preparations. (C) Cytofluorimetric analysis of FasL expression on purified melanosomes. Melanosomes were stained with anti-FasL NOK-1, anti-gp100, anti LAMP-2, and anti-CD63 mAbs, followed by incubation with FITC goat anti–mouse IgG (dark areas); an irrelevant isotype-matched Ab plus the FITC-conjugated goat anti–mouse IgG were used as negative control (clear areas). Fluorescence was analyzed by FACSCalibur™ and CELLQuest™ software. Melanosomes were detected as granules of the approximate mean size of 1 μm, relative to standard beads of 6 μm size.

    Journal: The Journal of Experimental Medicine

    Article Title: Induction of Lymphocyte Apoptosis by Tumor Cell Secretion of FasL-bearing Microvesicles

    doi: 10.1084/jem.20011624

    Figure Lengend Snippet: FasL expression in melanosomes purified from melanoma cells. (A) Western blot analysis of purified melanosome preparations. Western blot analysis was performed using proteins derived from melanosomal preparations purified from melanoma lines by sucrose gradient. Staining with G247 mAb revealed the presence in melanosome preparations of the 40–33 kD band pattern (lane 3–5). (B) Purity controls of melanosome preparations. Controls performed to check purity of melanosome preparations revealed the expression of the melanosomal marker gp100, and no significant contamination with Golgi, mitochondria, or plasma-membrane markers. The ER marker Bip/GRP78 was detectable in melanosome preparations. (C) Cytofluorimetric analysis of FasL expression on purified melanosomes. Melanosomes were stained with anti-FasL NOK-1, anti-gp100, anti LAMP-2, and anti-CD63 mAbs, followed by incubation with FITC goat anti–mouse IgG (dark areas); an irrelevant isotype-matched Ab plus the FITC-conjugated goat anti–mouse IgG were used as negative control (clear areas). Fluorescence was analyzed by FACSCalibur™ and CELLQuest™ software. Melanosomes were detected as granules of the approximate mean size of 1 μm, relative to standard beads of 6 μm size.

    Article Snippet: The red fluorescence of individual nuclei was measured with a FACSCalibur™ flow cytometer (Becton Dickinson).

    Techniques: Expressing, Purification, Western Blot, Derivative Assay, Staining, Marker, Incubation, Negative Control, Fluorescence, Software

    FasL intracellular localization in melanoma cells. (A) Cytofluorimetric analysis: melanoma cells were permeabilized with 70% methanol for 5 min on ice and subsequently stained with the anti-FasL G247 mAb (dark area) or with an isotype-matched IgG (clear area). Fluorescence was analyzed by FACSCalibur™ and CELLQuest™ software; data refer to three representative melanoma lines, from a panel of 10 tested. (B and C) Western blot detection of FasL in melanoma total cell lysates. Melanoma or PHA-activated Jurkat total cell lysates were stained by the anti-FasL G247 mAb. A significant positivity was detected in melanoma cell lysates (panel B, lanes 2–3), resembling the pattern observed in PHA-activated Jurkat cells (panel C, lane 2). Three major bands were detected in both melanoma and Jurkat cell lysates, corresponding to a molecular weight ranging between 40 and 33 kD and likely representing differently glycosylated forms of FasL molecule. (D) Western blot detection of FasL in cytoplasmic fractions of melanoma cells. Cytoplasmic-enriched preparations from melanoma cells were obtained as described in Materials and Methods. Western blot was stained with G247 mAb. Significant positivity (with bands ranging between 40 and 33 kD, with a predominance of the 40-kD band) was observed in both melanoma lines (lanes 2 and 3).

    Journal: The Journal of Experimental Medicine

    Article Title: Induction of Lymphocyte Apoptosis by Tumor Cell Secretion of FasL-bearing Microvesicles

    doi: 10.1084/jem.20011624

    Figure Lengend Snippet: FasL intracellular localization in melanoma cells. (A) Cytofluorimetric analysis: melanoma cells were permeabilized with 70% methanol for 5 min on ice and subsequently stained with the anti-FasL G247 mAb (dark area) or with an isotype-matched IgG (clear area). Fluorescence was analyzed by FACSCalibur™ and CELLQuest™ software; data refer to three representative melanoma lines, from a panel of 10 tested. (B and C) Western blot detection of FasL in melanoma total cell lysates. Melanoma or PHA-activated Jurkat total cell lysates were stained by the anti-FasL G247 mAb. A significant positivity was detected in melanoma cell lysates (panel B, lanes 2–3), resembling the pattern observed in PHA-activated Jurkat cells (panel C, lane 2). Three major bands were detected in both melanoma and Jurkat cell lysates, corresponding to a molecular weight ranging between 40 and 33 kD and likely representing differently glycosylated forms of FasL molecule. (D) Western blot detection of FasL in cytoplasmic fractions of melanoma cells. Cytoplasmic-enriched preparations from melanoma cells were obtained as described in Materials and Methods. Western blot was stained with G247 mAb. Significant positivity (with bands ranging between 40 and 33 kD, with a predominance of the 40-kD band) was observed in both melanoma lines (lanes 2 and 3).

    Article Snippet: The red fluorescence of individual nuclei was measured with a FACSCalibur™ flow cytometer (Becton Dickinson).

    Techniques: Staining, Fluorescence, Software, Western Blot, Molecular Weight

    FasL cannot be detected on melanoma cell membrane and in culture supernatant. (A) Cytofluorimetric analysis. Melanoma and transiently FasL-gene transfected 293 cells (control) were stained with anti-FasL NOK-1 mAb (dark area), or with an isotype-matched IgG (clear area), after 24 h-treatment with KB8301 10 μM; fluorescence was analyzed by FACSCalibur™ and CELLQuest™ software; data refer to three representative melanoma lines, from a panel of 10 tested. (B) Immunoprecipitation of membrane proteins: cell surface biotin-labeled cells of Me15392 were immunoprecipitated with G247 (lane 2) and NOK-1 (lane 3) anti-FasL mAb, with the isotype-matched murine IgG1 negative control (lane 4), or with the anti–HLA-class I mAb W6.32, as positive control. Cell lysates were analyzed in Western blot analysis with Streptavidin-HPR. A specific band (44 kD) is detectable only in lysates immunoprecipitated with W6.32 (lane 1). (C) Proapoptotic activity of melanoma cells. 51 [Cr]-labeled Jurkat cells (at 10 3 cells per well), in the presence or absence of neutralizing anti-Fas mAb ZB4 (50 ng/ml), were incubated for 16 h with either melanoma cells (10 5 cells per well), rFasL (100 ng/ml), or concentrated supernatant from 501mel culture (dilution 1:2). Killing was calculated as percentage of lysis. (D) Western blot analysis of FasL expression in supernatants from melanoma cell cultures. Melanoma supernantants were harvested from 72 h confluent cell cultures and concentrated by Centricon filter devices (cut-off 50 kD) (lanes 2 and 3). Concentrated supernatant from resting (lane 4) or PHA-activated (lane 5) Jurkat cells were additionally used as negative and positive controls, respectively. rFasL was also included (lane 1). Western blot analysis was stained with the anti-FasL G247 mAb. A 27-kD band, corresponding to soluble FasL, could be detected in supernatant from PHA-activated Jurkat cells.

    Journal: The Journal of Experimental Medicine

    Article Title: Induction of Lymphocyte Apoptosis by Tumor Cell Secretion of FasL-bearing Microvesicles

    doi: 10.1084/jem.20011624

    Figure Lengend Snippet: FasL cannot be detected on melanoma cell membrane and in culture supernatant. (A) Cytofluorimetric analysis. Melanoma and transiently FasL-gene transfected 293 cells (control) were stained with anti-FasL NOK-1 mAb (dark area), or with an isotype-matched IgG (clear area), after 24 h-treatment with KB8301 10 μM; fluorescence was analyzed by FACSCalibur™ and CELLQuest™ software; data refer to three representative melanoma lines, from a panel of 10 tested. (B) Immunoprecipitation of membrane proteins: cell surface biotin-labeled cells of Me15392 were immunoprecipitated with G247 (lane 2) and NOK-1 (lane 3) anti-FasL mAb, with the isotype-matched murine IgG1 negative control (lane 4), or with the anti–HLA-class I mAb W6.32, as positive control. Cell lysates were analyzed in Western blot analysis with Streptavidin-HPR. A specific band (44 kD) is detectable only in lysates immunoprecipitated with W6.32 (lane 1). (C) Proapoptotic activity of melanoma cells. 51 [Cr]-labeled Jurkat cells (at 10 3 cells per well), in the presence or absence of neutralizing anti-Fas mAb ZB4 (50 ng/ml), were incubated for 16 h with either melanoma cells (10 5 cells per well), rFasL (100 ng/ml), or concentrated supernatant from 501mel culture (dilution 1:2). Killing was calculated as percentage of lysis. (D) Western blot analysis of FasL expression in supernatants from melanoma cell cultures. Melanoma supernantants were harvested from 72 h confluent cell cultures and concentrated by Centricon filter devices (cut-off 50 kD) (lanes 2 and 3). Concentrated supernatant from resting (lane 4) or PHA-activated (lane 5) Jurkat cells were additionally used as negative and positive controls, respectively. rFasL was also included (lane 1). Western blot analysis was stained with the anti-FasL G247 mAb. A 27-kD band, corresponding to soluble FasL, could be detected in supernatant from PHA-activated Jurkat cells.

    Article Snippet: The red fluorescence of individual nuclei was measured with a FACSCalibur™ flow cytometer (Becton Dickinson).

    Techniques: Transfection, Staining, Fluorescence, Software, Immunoprecipitation, Labeling, Negative Control, Positive Control, Western Blot, Activity Assay, Incubation, Lysis, Expressing

    The gating strategy for the characterization of the mechanism of cell death (representative FACS histograms) THP-1 cells were seeded into 24-well plates at 10 6 cells/mL and cultured for 24 h in serum free RPMI media alone (A), or in the presence of 150 mM NaGlu (B), 200 mM Arg·Glu (C), 200 mM NaCl (D). Following incubation, cells were stained with annexin V-FITC (FL-1 channel) and PI (FL-2 channel) and 10,000 cells were analyzed using a FACSCalibur flow cytometer. Data are displayed as representative quadrant analyses showing the pattern of cytotoxicity for each treatment group; in each case the lower left quadrant represents Annexin V-ve/PI-ve (viable) cells, the lower right quadrant represents annexin V + ve/PI-ve (early apoptotic) cells, the upper right quadrant represents annexin V + ve/PI + ve (late apoptotic) cells and the upper left quadrant represents annexin V-ve/PI + ve (necrotic) cells. The percentage of cells in each quadrant is indicated.

    Journal: Toxicology in Vitro

    Article Title: The effects of arginine glutamate, a promising excipient for protein formulation, on cell viability: Comparisons with NaCl

    doi: 10.1016/j.tiv.2016.02.002

    Figure Lengend Snippet: The gating strategy for the characterization of the mechanism of cell death (representative FACS histograms) THP-1 cells were seeded into 24-well plates at 10 6 cells/mL and cultured for 24 h in serum free RPMI media alone (A), or in the presence of 150 mM NaGlu (B), 200 mM Arg·Glu (C), 200 mM NaCl (D). Following incubation, cells were stained with annexin V-FITC (FL-1 channel) and PI (FL-2 channel) and 10,000 cells were analyzed using a FACSCalibur flow cytometer. Data are displayed as representative quadrant analyses showing the pattern of cytotoxicity for each treatment group; in each case the lower left quadrant represents Annexin V-ve/PI-ve (viable) cells, the lower right quadrant represents annexin V + ve/PI-ve (early apoptotic) cells, the upper right quadrant represents annexin V + ve/PI + ve (late apoptotic) cells and the upper left quadrant represents annexin V-ve/PI + ve (necrotic) cells. The percentage of cells in each quadrant is indicated.

    Article Snippet: Cells (104 ) were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) and FlowJo software (Tree Star Inc., Ashland, OR, USA).

    Techniques: FACS, Cell Culture, Incubation, Staining, Flow Cytometry, Cytometry

    Characterization of the mechanism of cell death THP-1 cells were seeded into 24-well plates at 10 6 cells/mL in serum free RPMI media and were cultured for 4 h (A–D) or 24 h (E–H) at 37 °C in the presence of 50–200 mM NaCl (○), Arg·Glu (●), Arg·HCl (■) or NaGlu (∆). Control cells were cultured with medium alone. To characterize the extent and pattern of induced cytotoxicity, cells were stained with Annexin V-FITC (FL-1 channel) and PI (FL-2 channel) and 10,000 cells were analyzed using a FACSCalibur flow cytometer. The results are displayed as the percentages of cells that are Annexin V-ve/PI-ve (viable) (A, E), Annexin V + ve/PI-ve (early apoptotic) (B, F), Annexin V + ve/PI + ve (late apoptotic) (C, G), Annexin V-ve/PI + ve (necrotic) (D, H). In each case, data are shown as % total cells in each category (mean and SE for n = 3 experiments, where for clarity SE > 2% only are shown). The statistical significance of differences between cells cultured in medium alone and cells treated with various concentrations of salts was assessed by one way ANOVA (p

    Journal: Toxicology in Vitro

    Article Title: The effects of arginine glutamate, a promising excipient for protein formulation, on cell viability: Comparisons with NaCl

    doi: 10.1016/j.tiv.2016.02.002

    Figure Lengend Snippet: Characterization of the mechanism of cell death THP-1 cells were seeded into 24-well plates at 10 6 cells/mL in serum free RPMI media and were cultured for 4 h (A–D) or 24 h (E–H) at 37 °C in the presence of 50–200 mM NaCl (○), Arg·Glu (●), Arg·HCl (■) or NaGlu (∆). Control cells were cultured with medium alone. To characterize the extent and pattern of induced cytotoxicity, cells were stained with Annexin V-FITC (FL-1 channel) and PI (FL-2 channel) and 10,000 cells were analyzed using a FACSCalibur flow cytometer. The results are displayed as the percentages of cells that are Annexin V-ve/PI-ve (viable) (A, E), Annexin V + ve/PI-ve (early apoptotic) (B, F), Annexin V + ve/PI + ve (late apoptotic) (C, G), Annexin V-ve/PI + ve (necrotic) (D, H). In each case, data are shown as % total cells in each category (mean and SE for n = 3 experiments, where for clarity SE > 2% only are shown). The statistical significance of differences between cells cultured in medium alone and cells treated with various concentrations of salts was assessed by one way ANOVA (p

    Article Snippet: Cells (104 ) were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) and FlowJo software (Tree Star Inc., Ashland, OR, USA).

    Techniques: Cell Culture, Staining, Flow Cytometry, Cytometry

    Effects of Arg·Glu and other salts on CD54 expression and IL-8 production THP-1 cells were seeded into 24-well plates at 10 6 cells/mL in serum free RPMI media and were cultured for 24 h at 37 °C with medium alone or in the presence of 50–200 mM of Arg·Glu, NaCl, Arg·HCl or NaGlu. Positive control cells were cultured with LPS (0.1 μg/mL). At the end of the culture period, cells were stained for expression of CD54 or with isotype control (mouse IgG1κ) antibody and dead cells were excluded with PI. Data (10,000 cells) were acquired using a FACSCalibur flow cytometer and results are displayed as percentage positive cells (A) and mean fluorescence intensity (arbitrary units; au) (B) (mean and SE for six independent experiments). Following culture, samples were centrifuged at 1000 rpm for 5 min and supernatants were collected and analyzed for IL-8 content by ELISA (C). Results are displayed as mean and SEM of 3 independent experiments. The statistical significance of differences between cells cultured in medium alone and cells treated with various concentrations of salts or LPS was assessed by one way ANOVA (*p

    Journal: Toxicology in Vitro

    Article Title: The effects of arginine glutamate, a promising excipient for protein formulation, on cell viability: Comparisons with NaCl

    doi: 10.1016/j.tiv.2016.02.002

    Figure Lengend Snippet: Effects of Arg·Glu and other salts on CD54 expression and IL-8 production THP-1 cells were seeded into 24-well plates at 10 6 cells/mL in serum free RPMI media and were cultured for 24 h at 37 °C with medium alone or in the presence of 50–200 mM of Arg·Glu, NaCl, Arg·HCl or NaGlu. Positive control cells were cultured with LPS (0.1 μg/mL). At the end of the culture period, cells were stained for expression of CD54 or with isotype control (mouse IgG1κ) antibody and dead cells were excluded with PI. Data (10,000 cells) were acquired using a FACSCalibur flow cytometer and results are displayed as percentage positive cells (A) and mean fluorescence intensity (arbitrary units; au) (B) (mean and SE for six independent experiments). Following culture, samples were centrifuged at 1000 rpm for 5 min and supernatants were collected and analyzed for IL-8 content by ELISA (C). Results are displayed as mean and SEM of 3 independent experiments. The statistical significance of differences between cells cultured in medium alone and cells treated with various concentrations of salts or LPS was assessed by one way ANOVA (*p

    Article Snippet: Cells (104 ) were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) and FlowJo software (Tree Star Inc., Ashland, OR, USA).

    Techniques: Expressing, Cell Culture, Positive Control, Staining, Flow Cytometry, Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay

    Effect of changes in osmolality on THP-1 cell or fibroblast viability: comparisons of different salts THP-1 cells were seeded into 24-well plates at 10 6 cells/mL in serum free RPMI media (A, B) or human primary fibroblasts were seeded at 2 × 10 5 cells/mL in complete DMEM medium in flat-bottomed 24 well plates for 6 h at 37 °C (C, D). Cells were cultured in serum free media for 24 h at 37 °C with varying concentrations of NaCl (○), Arg·Glu (●), Arg·HCl (■) and NaGlu (∆) spanning a range of osmolalities from 280 to 525 mOsm/kg (A) or from 280 to 625 mOsm/kg (C) and a range of concentrations from 0 to 250 mM (B) or from 0 to 325 mM (D). Control cells were cultured with medium alone (280 mOsm/kg). Following culture, fibroblasts were trypsinized and THP-1 and fibrobalsts were harvested and cell viability was determined using PI. Cells (10,000) were analyzed by flow cytometry using a FACSCalibur flow cytometer for PI staining (FL2 channel; viability) against forward scatter (FSC; size). Data are displayed as % cell viability for n = 3–6 experiments (mean and SE) versus excipient with respect to cumulative osmolality (A, C) and the concentrations of each excipient required to achieve the required osmolality (B, D). The statistical significance of differences between cells cultured in medium alone (isotonicity) and cells treated with various concentrations of salts was assessed by one way ANOVA (p

    Journal: Toxicology in Vitro

    Article Title: The effects of arginine glutamate, a promising excipient for protein formulation, on cell viability: Comparisons with NaCl

    doi: 10.1016/j.tiv.2016.02.002

    Figure Lengend Snippet: Effect of changes in osmolality on THP-1 cell or fibroblast viability: comparisons of different salts THP-1 cells were seeded into 24-well plates at 10 6 cells/mL in serum free RPMI media (A, B) or human primary fibroblasts were seeded at 2 × 10 5 cells/mL in complete DMEM medium in flat-bottomed 24 well plates for 6 h at 37 °C (C, D). Cells were cultured in serum free media for 24 h at 37 °C with varying concentrations of NaCl (○), Arg·Glu (●), Arg·HCl (■) and NaGlu (∆) spanning a range of osmolalities from 280 to 525 mOsm/kg (A) or from 280 to 625 mOsm/kg (C) and a range of concentrations from 0 to 250 mM (B) or from 0 to 325 mM (D). Control cells were cultured with medium alone (280 mOsm/kg). Following culture, fibroblasts were trypsinized and THP-1 and fibrobalsts were harvested and cell viability was determined using PI. Cells (10,000) were analyzed by flow cytometry using a FACSCalibur flow cytometer for PI staining (FL2 channel; viability) against forward scatter (FSC; size). Data are displayed as % cell viability for n = 3–6 experiments (mean and SE) versus excipient with respect to cumulative osmolality (A, C) and the concentrations of each excipient required to achieve the required osmolality (B, D). The statistical significance of differences between cells cultured in medium alone (isotonicity) and cells treated with various concentrations of salts was assessed by one way ANOVA (p

    Article Snippet: Cells (104 ) were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) and FlowJo software (Tree Star Inc., Ashland, OR, USA).

    Techniques: Cell Culture, Flow Cytometry, Cytometry, Staining

    Synergistic cytotoxicity of HHT and ETP in AML cells. Notes: ( A ) THP1 and HL60 cells were treated with vehicle control (Ctrl), HHT, ETP, or in combination as indicated for 48 hours. Cell viability was determined via trypan blue exclusion assay. Results relative to Ctrl are shown (%). ( B, C ) HL60 cells were treated as in ( A ). ( B ) The percentage of apoptotic cells was determined by flow cytometry analysis using annexin-V/propidium iodide double staining. The statistical analysis is shown. ( C ) The protein expressions of cleaved caspase-9 and cleaved caspase-3 were measured by Western blot analysis. PARP was used as a loading control. ( D ) Primary AML cells from three patients (AML-1, AML-2, and AML-3) were treated as indicated for 48 hours. Cell viability was determined via trypan blue exclusion assay. Results relative to Ctrl are shown (%). ( E, F ) Primary AML cells (AML-2) were treated as in ( D ). ( E ) The percentage of apoptotic cells was determined by flow cytometry analysis using Annexin-V/propidium iodide double staining. The statistical analysis is shown. ( F ) The protein expressions of cleaved caspase-9 and cleaved caspase-3 were measured by Western blot analysis. PARP was used as a loading control. Data were obtained from at least three independent experiments and analyzed by Student’s t -test. Data are expressed as mean ± SD. ** P

    Journal: Cancer Management and Research

    Article Title: Synergistic cytotoxicity of homoharringtonine and etoposide in acute myeloid leukemia cells involves disrupted antioxidant defense

    doi: 10.2147/CMAR.S187597

    Figure Lengend Snippet: Synergistic cytotoxicity of HHT and ETP in AML cells. Notes: ( A ) THP1 and HL60 cells were treated with vehicle control (Ctrl), HHT, ETP, or in combination as indicated for 48 hours. Cell viability was determined via trypan blue exclusion assay. Results relative to Ctrl are shown (%). ( B, C ) HL60 cells were treated as in ( A ). ( B ) The percentage of apoptotic cells was determined by flow cytometry analysis using annexin-V/propidium iodide double staining. The statistical analysis is shown. ( C ) The protein expressions of cleaved caspase-9 and cleaved caspase-3 were measured by Western blot analysis. PARP was used as a loading control. ( D ) Primary AML cells from three patients (AML-1, AML-2, and AML-3) were treated as indicated for 48 hours. Cell viability was determined via trypan blue exclusion assay. Results relative to Ctrl are shown (%). ( E, F ) Primary AML cells (AML-2) were treated as in ( D ). ( E ) The percentage of apoptotic cells was determined by flow cytometry analysis using Annexin-V/propidium iodide double staining. The statistical analysis is shown. ( F ) The protein expressions of cleaved caspase-9 and cleaved caspase-3 were measured by Western blot analysis. PARP was used as a loading control. Data were obtained from at least three independent experiments and analyzed by Student’s t -test. Data are expressed as mean ± SD. ** P

    Article Snippet: The percentage of apoptotic cells was determined by flow cytometry analysis (FACSCalibur Flow Cytometer; BD Biosciences, San Jose, CA, USA).

    Techniques: Trypan Blue Exclusion Assay, Flow Cytometry, Cytometry, Double Staining, Western Blot

    HHT causes elevated ROS generation by disabling thioredoxin-mediated antioxidant defense. Notes: ( A ) HL60, THP1, and primary AML cells (AML-2) were treated with vehicle control (Ctrl), HHT, ETP, or in combination as indicated for 48 hours. The protein expression of Trx1 was determined by Western blot. GAPDH was used as a loading control. The representative images (left) and the quantification of band intensity (right) are shown. ( B – D ) HL60 cells stably overexpressing vector or Trx1 were treated as in ( A ). ( B ) The protein expression of Trx1 was determined by Western blot. GAPDH was used as a loading control. The representative images (upper) and the quantification of band intensity (lower) are shown. ( C ) The intracellular ROS level was detected by flow cytometry analysis using DCFH-DA. Results relative to Ctrl are shown. ( D ) Cell viability was determined via trypan blue exclusion assay. Results relative to Ctrl are shown (%). Data were obtained from at least three independent experiments and analyzed by Student’s t -test. Data are expressed as mean ± SD. ** P

    Journal: Cancer Management and Research

    Article Title: Synergistic cytotoxicity of homoharringtonine and etoposide in acute myeloid leukemia cells involves disrupted antioxidant defense

    doi: 10.2147/CMAR.S187597

    Figure Lengend Snippet: HHT causes elevated ROS generation by disabling thioredoxin-mediated antioxidant defense. Notes: ( A ) HL60, THP1, and primary AML cells (AML-2) were treated with vehicle control (Ctrl), HHT, ETP, or in combination as indicated for 48 hours. The protein expression of Trx1 was determined by Western blot. GAPDH was used as a loading control. The representative images (left) and the quantification of band intensity (right) are shown. ( B – D ) HL60 cells stably overexpressing vector or Trx1 were treated as in ( A ). ( B ) The protein expression of Trx1 was determined by Western blot. GAPDH was used as a loading control. The representative images (upper) and the quantification of band intensity (lower) are shown. ( C ) The intracellular ROS level was detected by flow cytometry analysis using DCFH-DA. Results relative to Ctrl are shown. ( D ) Cell viability was determined via trypan blue exclusion assay. Results relative to Ctrl are shown (%). Data were obtained from at least three independent experiments and analyzed by Student’s t -test. Data are expressed as mean ± SD. ** P

    Article Snippet: The percentage of apoptotic cells was determined by flow cytometry analysis (FACSCalibur Flow Cytometer; BD Biosciences, San Jose, CA, USA).

    Techniques: Expressing, Western Blot, Stable Transfection, Plasmid Preparation, Flow Cytometry, Cytometry, Trypan Blue Exclusion Assay

    HHT causes elevated ROS generation in AML cells treated with ETP. Notes: HL60 cells ( A ) and THP1 cells ( B ) were treated with vehicle control (Ctrl), HHT, ETP, or in combination as indicated for 48 hours. The intracellular ROS level was detected by flow cytometry analysis using DCFH-DA. Results relative to Ctrl are shown. ( C ) The primary AML cells (AML-2) were treated as indicated for 48 hours. The intracellular ROS level was detected by flow cytometry analysis using DCFH-DA. Results relative to Ctrl are shown. Data were obtained from at least three independent experiments and analyzed by Student’s t -test. Data are expressed as mean ± SD. ** P

    Journal: Cancer Management and Research

    Article Title: Synergistic cytotoxicity of homoharringtonine and etoposide in acute myeloid leukemia cells involves disrupted antioxidant defense

    doi: 10.2147/CMAR.S187597

    Figure Lengend Snippet: HHT causes elevated ROS generation in AML cells treated with ETP. Notes: HL60 cells ( A ) and THP1 cells ( B ) were treated with vehicle control (Ctrl), HHT, ETP, or in combination as indicated for 48 hours. The intracellular ROS level was detected by flow cytometry analysis using DCFH-DA. Results relative to Ctrl are shown. ( C ) The primary AML cells (AML-2) were treated as indicated for 48 hours. The intracellular ROS level was detected by flow cytometry analysis using DCFH-DA. Results relative to Ctrl are shown. Data were obtained from at least three independent experiments and analyzed by Student’s t -test. Data are expressed as mean ± SD. ** P

    Article Snippet: The percentage of apoptotic cells was determined by flow cytometry analysis (FACSCalibur Flow Cytometer; BD Biosciences, San Jose, CA, USA).

    Techniques: Flow Cytometry, Cytometry

    ROS scavenge diminishes HHT-augmented ETP cytotoxicity. Notes: ( A – C ) HL60 cells were treated with vehicle control (Ctrl), HHT, ETP, or in combination in the presence or absence of NAC as indicated for 48 hours. ( A ) The intracellular ROS level was detected by flow cytometry analysis using DCFH-DA. Results relative to Ctrl are shown. ( B ) Cell viability was determined via trypan blue exclusion assay. Results relative to Ctrl are shown (%). ( C ) The protein expressions of cleaved caspase-9 and cleaved caspase-3 were measured by Western blot analysis. PARP was used as a loading control. ( D – F ) Primary AML cells (AML-2) were treated as in ( A ). The intracellular ROS level ( D ), cell viability ( E ), and protein expressions of cleaved caspase-9 and cleaved caspase-3 ( F ) were determined as in ( A – C ). Data were obtained from at least three independent experiments and analyzed by Student’s t -test. Data are expressed as mean ± SD. ** P

    Journal: Cancer Management and Research

    Article Title: Synergistic cytotoxicity of homoharringtonine and etoposide in acute myeloid leukemia cells involves disrupted antioxidant defense

    doi: 10.2147/CMAR.S187597

    Figure Lengend Snippet: ROS scavenge diminishes HHT-augmented ETP cytotoxicity. Notes: ( A – C ) HL60 cells were treated with vehicle control (Ctrl), HHT, ETP, or in combination in the presence or absence of NAC as indicated for 48 hours. ( A ) The intracellular ROS level was detected by flow cytometry analysis using DCFH-DA. Results relative to Ctrl are shown. ( B ) Cell viability was determined via trypan blue exclusion assay. Results relative to Ctrl are shown (%). ( C ) The protein expressions of cleaved caspase-9 and cleaved caspase-3 were measured by Western blot analysis. PARP was used as a loading control. ( D – F ) Primary AML cells (AML-2) were treated as in ( A ). The intracellular ROS level ( D ), cell viability ( E ), and protein expressions of cleaved caspase-9 and cleaved caspase-3 ( F ) were determined as in ( A – C ). Data were obtained from at least three independent experiments and analyzed by Student’s t -test. Data are expressed as mean ± SD. ** P

    Article Snippet: The percentage of apoptotic cells was determined by flow cytometry analysis (FACSCalibur Flow Cytometer; BD Biosciences, San Jose, CA, USA).

    Techniques: Flow Cytometry, Cytometry, Trypan Blue Exclusion Assay, Western Blot

    Depletion of thioredoxin sensitizes AML to ETP treatment. Notes: ( A – C ) HL60 cells transfected with siCtrl or siTrx1 were treated with vehicle control (Ctrl) or 2 µM ETP for 48 hours. ( A ) The protein expression of Trx1 was determined by Western blot. GAPDH was used as a loading control. The representative images (left) and the quantification of band intensity (right) are shown. ( B ) The intracellular ROS level was detected by flow cytometry analysis using DCFH-DA. Results relative to Ctrl are shown. ( C ) Cell viability was determined via trypan blue exclusion assay. Results relative to Ctrl are shown (%). Data were obtained from at least three independent experiments and analyzed by Student’s t -test. Data are presented as mean ± SD. ** P

    Journal: Cancer Management and Research

    Article Title: Synergistic cytotoxicity of homoharringtonine and etoposide in acute myeloid leukemia cells involves disrupted antioxidant defense

    doi: 10.2147/CMAR.S187597

    Figure Lengend Snippet: Depletion of thioredoxin sensitizes AML to ETP treatment. Notes: ( A – C ) HL60 cells transfected with siCtrl or siTrx1 were treated with vehicle control (Ctrl) or 2 µM ETP for 48 hours. ( A ) The protein expression of Trx1 was determined by Western blot. GAPDH was used as a loading control. The representative images (left) and the quantification of band intensity (right) are shown. ( B ) The intracellular ROS level was detected by flow cytometry analysis using DCFH-DA. Results relative to Ctrl are shown. ( C ) Cell viability was determined via trypan blue exclusion assay. Results relative to Ctrl are shown (%). Data were obtained from at least three independent experiments and analyzed by Student’s t -test. Data are presented as mean ± SD. ** P

    Article Snippet: The percentage of apoptotic cells was determined by flow cytometry analysis (FACSCalibur Flow Cytometer; BD Biosciences, San Jose, CA, USA).

    Techniques: Transfection, Expressing, Western Blot, Flow Cytometry, Cytometry, Trypan Blue Exclusion Assay

    GSC-derived exosomes are internalized by monocytes and stimulate proliferation of CD14 negatively-sorted PBMCs. Fluorescent exosomes (A) were incubated with PBMCs for 5 hours and the uptake by CD14+ monocytes, CD4+ or CD8+ T cells was measured by flow cytometric analysis in the bulk population. In representative cytometry histograms, the isotype control is in black and in grey are PBMCs cells incubated with labelled-exosomes and gated on CD14+ monocytes (left, top), on CD4+ (left, bottom) or on CD8+ T cells (right, bottom). (B) Gating and sorting strategies of PBMCs and CD14-depleted PBMC. (Top, left) physical parameters, i.e. forward scatter (FSC) and side scatter (SSC), were used to select PBMCs (gate R1). Monocytes were recognized by evaluating, in PBMCs, the expression of CD14 (gate R2, top, right panel). A PE-isotype matched antibody was used to define R2 (top, central panel). PBMCs-depleted cells were identified as cells included in R1 but not in R2. (C) Representative dot-plots showing the reanalysis of the FACS-sorted PBMCs (left panel) and CD14-depleted PBMCs (right panel). As expected, CD14-positive cells were present in the sorted PBMCs- but not in the CD14-depleted- samples. (D) Proliferation of both unfractioned PBMCs and PBMCs depleted of the CD14+ population (CD14-) was measured, after CFSE labelling assay, by flow cytometry. Cells were pre-incubated without (white column, CTRL) or with (black column, GSC-EXO) GSC-derived exosomes. Columns, mean (n = 6); bars, SD; *, significantly different from the control; P

    Journal: PLoS ONE

    Article Title: Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells

    doi: 10.1371/journal.pone.0169932

    Figure Lengend Snippet: GSC-derived exosomes are internalized by monocytes and stimulate proliferation of CD14 negatively-sorted PBMCs. Fluorescent exosomes (A) were incubated with PBMCs for 5 hours and the uptake by CD14+ monocytes, CD4+ or CD8+ T cells was measured by flow cytometric analysis in the bulk population. In representative cytometry histograms, the isotype control is in black and in grey are PBMCs cells incubated with labelled-exosomes and gated on CD14+ monocytes (left, top), on CD4+ (left, bottom) or on CD8+ T cells (right, bottom). (B) Gating and sorting strategies of PBMCs and CD14-depleted PBMC. (Top, left) physical parameters, i.e. forward scatter (FSC) and side scatter (SSC), were used to select PBMCs (gate R1). Monocytes were recognized by evaluating, in PBMCs, the expression of CD14 (gate R2, top, right panel). A PE-isotype matched antibody was used to define R2 (top, central panel). PBMCs-depleted cells were identified as cells included in R1 but not in R2. (C) Representative dot-plots showing the reanalysis of the FACS-sorted PBMCs (left panel) and CD14-depleted PBMCs (right panel). As expected, CD14-positive cells were present in the sorted PBMCs- but not in the CD14-depleted- samples. (D) Proliferation of both unfractioned PBMCs and PBMCs depleted of the CD14+ population (CD14-) was measured, after CFSE labelling assay, by flow cytometry. Cells were pre-incubated without (white column, CTRL) or with (black column, GSC-EXO) GSC-derived exosomes. Columns, mean (n = 6); bars, SD; *, significantly different from the control; P

    Article Snippet: Purity of monocytes was over 95% as proved by staining with anti-CD14 mAb (eBiosciences) and flow cytometry analysis (FACScalibur cytometer, BD, Biosciences).

    Techniques: Derivative Assay, Incubation, Flow Cytometry, Cytometry, Expressing, FACS

    GSC-derived exosomes stimulate IL-1β, IL-6 and IL-10 production in unstimulated CD14+ monocytes within PBMC population. Unstimulated PBMCs were incubated in the absence (white column, CTRL) or presence (black column, GSC-EXO) of GSC-derived exosomes. Incubation with LPS (square column) was used as monocyte stimulation positive control. Cells were surface stained with anti-CD14 and then stained to detect an intracellular level of IL-1β, IL-6 and IL-10 by flow cytometry. (A-C) Representative FACS plot of the intracellular staining is shown by the indicated percentage of CD14+/IL-1β+, CD14+/IL-6+ and CD14+/IL-10+ positive cells, respectively. (D-F) The mean of the experiments is shown (n = 6); bars, SD; *, significantly different from the control; P

    Journal: PLoS ONE

    Article Title: Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells

    doi: 10.1371/journal.pone.0169932

    Figure Lengend Snippet: GSC-derived exosomes stimulate IL-1β, IL-6 and IL-10 production in unstimulated CD14+ monocytes within PBMC population. Unstimulated PBMCs were incubated in the absence (white column, CTRL) or presence (black column, GSC-EXO) of GSC-derived exosomes. Incubation with LPS (square column) was used as monocyte stimulation positive control. Cells were surface stained with anti-CD14 and then stained to detect an intracellular level of IL-1β, IL-6 and IL-10 by flow cytometry. (A-C) Representative FACS plot of the intracellular staining is shown by the indicated percentage of CD14+/IL-1β+, CD14+/IL-6+ and CD14+/IL-10+ positive cells, respectively. (D-F) The mean of the experiments is shown (n = 6); bars, SD; *, significantly different from the control; P

    Article Snippet: Purity of monocytes was over 95% as proved by staining with anti-CD14 mAb (eBiosciences) and flow cytometry analysis (FACScalibur cytometer, BD, Biosciences).

    Techniques: Derivative Assay, Incubation, Positive Control, Staining, Flow Cytometry, Cytometry, FACS

    Within PBMCs population, GSC-derived exosomes promote an immunosuppressive phenotype in monocytes and stimulate the production of arginase-1 and IL-10 by Mo-MDSCs. Unstimulated PBMCs were incubated in absence (CTRL) or presence (GSC-EXO) of GSC-derived exosomes. Cells were surface stained with anti-CD14, anti CD33, anti CD11b and HLA-DR and then stained to detect intracellular level of IL-10 and arginase-1 by flow cytometry. (A) Gating strategy: physical parameters, i.e. forward scatter (FSC) and side scatter (SSC), were used to select monocytes (gate R3, left panel). Monocytes were recognized evaluating the expression of CD11b/CD33 (gate R4, middle panel) and CD14/HLA-DR (gate R5, right panel). (B) Representative FACS histograms of the intracellular staining of IL-10 and arginase-1 and of HLA-DR staining of CD14+/CD11b/CD33+ cells are shown. (C) The percentage of cells expressing IL-10 and arginase-1 and the MFI ratio of HLA-DR expression are shown (n = 6); bars, SD;*, significantly different from the control; P

    Journal: PLoS ONE

    Article Title: Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells

    doi: 10.1371/journal.pone.0169932

    Figure Lengend Snippet: Within PBMCs population, GSC-derived exosomes promote an immunosuppressive phenotype in monocytes and stimulate the production of arginase-1 and IL-10 by Mo-MDSCs. Unstimulated PBMCs were incubated in absence (CTRL) or presence (GSC-EXO) of GSC-derived exosomes. Cells were surface stained with anti-CD14, anti CD33, anti CD11b and HLA-DR and then stained to detect intracellular level of IL-10 and arginase-1 by flow cytometry. (A) Gating strategy: physical parameters, i.e. forward scatter (FSC) and side scatter (SSC), were used to select monocytes (gate R3, left panel). Monocytes were recognized evaluating the expression of CD11b/CD33 (gate R4, middle panel) and CD14/HLA-DR (gate R5, right panel). (B) Representative FACS histograms of the intracellular staining of IL-10 and arginase-1 and of HLA-DR staining of CD14+/CD11b/CD33+ cells are shown. (C) The percentage of cells expressing IL-10 and arginase-1 and the MFI ratio of HLA-DR expression are shown (n = 6); bars, SD;*, significantly different from the control; P

    Article Snippet: Purity of monocytes was over 95% as proved by staining with anti-CD14 mAb (eBiosciences) and flow cytometry analysis (FACScalibur cytometer, BD, Biosciences).

    Techniques: Derivative Assay, Incubation, Staining, Flow Cytometry, Cytometry, Expressing, FACS

    GSC-derived exosomes stimulate CD25 expression and proliferation of isolated CD4+ T cells but do not affect differentiation and suppressive activity of Treg cells. CD4+ T cells, isolated from PBMCs by negative selection, were stimulated with anti-CD3, anti-CD28 and IL-2 in the absence (white column, CTRL) or presence (black column, GSC-EXO) of GSC-derived exosomes. Expression of CD25 (A), percentage of proliferative CFSE-labelled cells in the presence of indicated stimuli (B) and frequency of CD4+/CD25+/FoxP3+ (C) was determined by flow cytometry on day 4. Columns, mean (n = 6); bars, SD; *, significantly different from the control; p

    Journal: PLoS ONE

    Article Title: Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells

    doi: 10.1371/journal.pone.0169932

    Figure Lengend Snippet: GSC-derived exosomes stimulate CD25 expression and proliferation of isolated CD4+ T cells but do not affect differentiation and suppressive activity of Treg cells. CD4+ T cells, isolated from PBMCs by negative selection, were stimulated with anti-CD3, anti-CD28 and IL-2 in the absence (white column, CTRL) or presence (black column, GSC-EXO) of GSC-derived exosomes. Expression of CD25 (A), percentage of proliferative CFSE-labelled cells in the presence of indicated stimuli (B) and frequency of CD4+/CD25+/FoxP3+ (C) was determined by flow cytometry on day 4. Columns, mean (n = 6); bars, SD; *, significantly different from the control; p

    Article Snippet: Purity of monocytes was over 95% as proved by staining with anti-CD14 mAb (eBiosciences) and flow cytometry analysis (FACScalibur cytometer, BD, Biosciences).

    Techniques: Derivative Assay, Expressing, Isolation, Activity Assay, Selection, Flow Cytometry, Cytometry

    GSC-derived exosomes inhibit T-cell proliferation and expression of activation markers and modulate cytokine production of PBMCs. CFSE-labeled PBMCs isolated from healthy donors were pretreated for 24 hours without (white column, CTRL) or with GSC-derived exosomes (black column, GSC-EXO) and stimulated for 4 days with anti-CD3 and anti-CD28. (A) Representative microscope images and respective cytometry CFSE histograms, showing the fraction of proliferative CD3+ T cells, in unstimulated PBMCs (i), stimulated PBMCs (ii) and exosomes-treated stimulated PBMCs (iii). (B-D) Histograms showing, within the PBMCs, the fraction of proliferating CD3+ (B), CD4+ (C) and CD8+ (D) T cells. (E-F) CD3+ T-cell expression of CD25 and CD69 was measured by flow cytometry on day 2. (G-L) PBMC-derived supernatants were harvested after 48 hours and used for ELISA with the Bio-plex cytokine assay system. Cytokines that showed statistically significant differences with the exosome treatment are reported. Concentration of IL-2 (G), INF-γ (H), TNF-α (I) and IL-5 (J) are expressed as pg/ml. In B-L, the data are presented as mean ± SD (n = 6). *, p

    Journal: PLoS ONE

    Article Title: Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells

    doi: 10.1371/journal.pone.0169932

    Figure Lengend Snippet: GSC-derived exosomes inhibit T-cell proliferation and expression of activation markers and modulate cytokine production of PBMCs. CFSE-labeled PBMCs isolated from healthy donors were pretreated for 24 hours without (white column, CTRL) or with GSC-derived exosomes (black column, GSC-EXO) and stimulated for 4 days with anti-CD3 and anti-CD28. (A) Representative microscope images and respective cytometry CFSE histograms, showing the fraction of proliferative CD3+ T cells, in unstimulated PBMCs (i), stimulated PBMCs (ii) and exosomes-treated stimulated PBMCs (iii). (B-D) Histograms showing, within the PBMCs, the fraction of proliferating CD3+ (B), CD4+ (C) and CD8+ (D) T cells. (E-F) CD3+ T-cell expression of CD25 and CD69 was measured by flow cytometry on day 2. (G-L) PBMC-derived supernatants were harvested after 48 hours and used for ELISA with the Bio-plex cytokine assay system. Cytokines that showed statistically significant differences with the exosome treatment are reported. Concentration of IL-2 (G), INF-γ (H), TNF-α (I) and IL-5 (J) are expressed as pg/ml. In B-L, the data are presented as mean ± SD (n = 6). *, p

    Article Snippet: Purity of monocytes was over 95% as proved by staining with anti-CD14 mAb (eBiosciences) and flow cytometry analysis (FACScalibur cytometer, BD, Biosciences).

    Techniques: Derivative Assay, Expressing, Activation Assay, Labeling, Isolation, Microscopy, Cytometry, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cytokine Assay, Concentration Assay

    Detection of subsets of CD3 + CD20 + T cells in healthy subjects . (A) (i) Typical flow cytometry dot plots of the forward side scatter (FSC) against sideward scatter (SSC) of peripheral blood lymphocytes (PBLs). Within the lymphocyte population, CD3-FITC/CD19-APC/CD20-PE triple-staining was performed on the cells. This staining enabled visualization of (ii) double-positive CD3 + CD20 + , (iii) CD3 + CD19 - , and (iv) CD19 + CD20 + events. (B) PBLs expressing a CD3 + phenotype (α-CD3-FITC) were gated and evaluated for their expression patterns of CD19 (α-CD19-APC) and CD20 (α-CD20-PE). Alternatively, PBLs were stained with α-CD4-FITC or αCD8-FITC and α-CD19-APC/α-CD20-PE to determine the percentage of CD4 + /CD20 + /CD19 - and CD8 + /CD20 + /CD19 - cells. (C) Lymphocytes were stained with α-CD3-Pacific blue/α-CD19-APC/α-CD20-FITC, whereupon α-CD3 + /α-CD19-/α-CD20 + and α-CD3 + /α-CD19 - /α-CD20 - cells were sorted as illustrated with the sort plots. Subsequently, sorted populations were stained with a mix of α-CD4-PE/α-CD8-PE and the respective populations were analyzed by confocal microscopy for CD4/CD8 (red pseudo color) and CD20 (green pseudo color). Confocal microscopy was performed with a Leica DM IRE2 Inverted microscope (objectives: HCX PL APO 63 ×/1.3 with glycerin; camera: Stanford Photonics XR/Mega-10I (intensified) charge-coupled device (CCD) camera; software: Yokogawa Confocal Scanner Unit CSU10). This experiment was performed on four occasions with similar results. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

    Journal: Arthritis Research & Therapy

    Article Title: Frequency of Th17 CD20+ cells in the peripheral blood of rheumatoid arthritis patients is higher compared to healthy subjects

    doi: 10.1186/ar3541

    Figure Lengend Snippet: Detection of subsets of CD3 + CD20 + T cells in healthy subjects . (A) (i) Typical flow cytometry dot plots of the forward side scatter (FSC) against sideward scatter (SSC) of peripheral blood lymphocytes (PBLs). Within the lymphocyte population, CD3-FITC/CD19-APC/CD20-PE triple-staining was performed on the cells. This staining enabled visualization of (ii) double-positive CD3 + CD20 + , (iii) CD3 + CD19 - , and (iv) CD19 + CD20 + events. (B) PBLs expressing a CD3 + phenotype (α-CD3-FITC) were gated and evaluated for their expression patterns of CD19 (α-CD19-APC) and CD20 (α-CD20-PE). Alternatively, PBLs were stained with α-CD4-FITC or αCD8-FITC and α-CD19-APC/α-CD20-PE to determine the percentage of CD4 + /CD20 + /CD19 - and CD8 + /CD20 + /CD19 - cells. (C) Lymphocytes were stained with α-CD3-Pacific blue/α-CD19-APC/α-CD20-FITC, whereupon α-CD3 + /α-CD19-/α-CD20 + and α-CD3 + /α-CD19 - /α-CD20 - cells were sorted as illustrated with the sort plots. Subsequently, sorted populations were stained with a mix of α-CD4-PE/α-CD8-PE and the respective populations were analyzed by confocal microscopy for CD4/CD8 (red pseudo color) and CD20 (green pseudo color). Confocal microscopy was performed with a Leica DM IRE2 Inverted microscope (objectives: HCX PL APO 63 ×/1.3 with glycerin; camera: Stanford Photonics XR/Mega-10I (intensified) charge-coupled device (CCD) camera; software: Yokogawa Confocal Scanner Unit CSU10). This experiment was performed on four occasions with similar results. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

    Article Snippet: To confirm that the anti-CD20-conjugated antibodies were binding specifically to surface-expressed CD20 antigen, isolated PMBCs were washed and pre-incubated with 5 μg/mL RTX (Hoffman-La Roche, Basel, Switzerland) on ice for 15 minutes and washed again before beginning the staining procedures for flow cytometry analysis (Accuri flow cytometer; BD Biosciences, San Jose, CA, USA).

    Techniques: Flow Cytometry, Cytometry, Staining, Expressing, Confocal Microscopy, Inverted Microscopy, Software

    Expression of CD20 on B and T lymphocytes from the peripheral blood of healthy subjects (HS) compared with rheumatoid arthritis (RA) patients, as obtained by flow cytometry . (A) The percentage of CD20 + peripheral blood lymphocytes in HS and RA patients. (B) The percentage CD3 + CD20 + T cells in HS and RA patients. (C) The percentage of CD19 + CD20 + B cells in HS and RA patients as analyzed by flow cytometry. The bars represent the median percentage and interquartile range. The expression patterns of CD20 + cells represent data from 11 HS and 8 RA blood samples in total.

    Journal: Arthritis Research & Therapy

    Article Title: Frequency of Th17 CD20+ cells in the peripheral blood of rheumatoid arthritis patients is higher compared to healthy subjects

    doi: 10.1186/ar3541

    Figure Lengend Snippet: Expression of CD20 on B and T lymphocytes from the peripheral blood of healthy subjects (HS) compared with rheumatoid arthritis (RA) patients, as obtained by flow cytometry . (A) The percentage of CD20 + peripheral blood lymphocytes in HS and RA patients. (B) The percentage CD3 + CD20 + T cells in HS and RA patients. (C) The percentage of CD19 + CD20 + B cells in HS and RA patients as analyzed by flow cytometry. The bars represent the median percentage and interquartile range. The expression patterns of CD20 + cells represent data from 11 HS and 8 RA blood samples in total.

    Article Snippet: To confirm that the anti-CD20-conjugated antibodies were binding specifically to surface-expressed CD20 antigen, isolated PMBCs were washed and pre-incubated with 5 μg/mL RTX (Hoffman-La Roche, Basel, Switzerland) on ice for 15 minutes and washed again before beginning the staining procedures for flow cytometry analysis (Accuri flow cytometer; BD Biosciences, San Jose, CA, USA).

    Techniques: Expressing, Flow Cytometry, Cytometry

    CD20 + , IL-17-secreting, and IL-17-secreting/CD20 + lymphocytes in healthy subjects (HS) and rheumatoid arthritis (RA) patients . (A) Dot plot of size versus CD20 positivity in representative peripheral blood lymphocyte (PBL) samples from an HS and an RA patient. (B) Typical flow cytometry dot plots showing the proportion of CD20 + and IL-17-secreting lymphocytes in representative PBL samples from an HS (left panel) and an RA patient (right panel). Each dot plot shows the proportion of CD20 + cells (lower right quadrant), IL-17-secreting cells (upper left quadrant), and IL-17-secreting CD20 + cells (upper right quadrant). (C) The proportion of PBLs that were IL-17-secreting in HS ( n = 6) and RA subjects ( n = 9, left panel) and the proportion of IL-17-secreting cells that were specifically CD20 + T cells (right panel). The horizontal bars represent the median percentage and interquartile range of each group. IL, interleukin.

    Journal: Arthritis Research & Therapy

    Article Title: Frequency of Th17 CD20+ cells in the peripheral blood of rheumatoid arthritis patients is higher compared to healthy subjects

    doi: 10.1186/ar3541

    Figure Lengend Snippet: CD20 + , IL-17-secreting, and IL-17-secreting/CD20 + lymphocytes in healthy subjects (HS) and rheumatoid arthritis (RA) patients . (A) Dot plot of size versus CD20 positivity in representative peripheral blood lymphocyte (PBL) samples from an HS and an RA patient. (B) Typical flow cytometry dot plots showing the proportion of CD20 + and IL-17-secreting lymphocytes in representative PBL samples from an HS (left panel) and an RA patient (right panel). Each dot plot shows the proportion of CD20 + cells (lower right quadrant), IL-17-secreting cells (upper left quadrant), and IL-17-secreting CD20 + cells (upper right quadrant). (C) The proportion of PBLs that were IL-17-secreting in HS ( n = 6) and RA subjects ( n = 9, left panel) and the proportion of IL-17-secreting cells that were specifically CD20 + T cells (right panel). The horizontal bars represent the median percentage and interquartile range of each group. IL, interleukin.

    Article Snippet: To confirm that the anti-CD20-conjugated antibodies were binding specifically to surface-expressed CD20 antigen, isolated PMBCs were washed and pre-incubated with 5 μg/mL RTX (Hoffman-La Roche, Basel, Switzerland) on ice for 15 minutes and washed again before beginning the staining procedures for flow cytometry analysis (Accuri flow cytometer; BD Biosciences, San Jose, CA, USA).

    Techniques: Flow Cytometry, Cytometry

    Gating and control rationale for CD20 + T-cell selection . (A) (i) Typical flow cytometry dot plot of the forward and side scatter of peripheral blood mononuclear cells (PBMCs). (ii) Dot plot of P1 gated cells (as indicated in A) stained with CD20-FITC and CD19-PE and shown in 'R2'. (iii) Dot plot of P1 gated cells, except 'R2', stained with CD20-FITC and CD3-CyQ. (B) (i) Typical flow cytometry dot plot of the forward and side scatter of PBMCs. (ii) Dot plot of P1 gated cells stained with isotype control IgG-FITC and isotype control IgG-PE and shown in 'R2' (iii). Dot plot of P1 gated cells, except 'R2', stained with isotype control IgG-FITC and isotype control CD3-CyQ. (C) (i) Typical flow cytometry dot plot of the forward and side scatter of PBMCs. (ii) Dot plot of P1 gated cells stained with CD20-FITC and CD19-PE post-RTX treatment and shown in 'R2'. (iii) Dot plot of P1 gated cells, except 'R2', stained with CD20-FITC and CD3-CyQ post-RTX incubation.

    Journal: Arthritis Research & Therapy

    Article Title: Frequency of Th17 CD20+ cells in the peripheral blood of rheumatoid arthritis patients is higher compared to healthy subjects

    doi: 10.1186/ar3541

    Figure Lengend Snippet: Gating and control rationale for CD20 + T-cell selection . (A) (i) Typical flow cytometry dot plot of the forward and side scatter of peripheral blood mononuclear cells (PBMCs). (ii) Dot plot of P1 gated cells (as indicated in A) stained with CD20-FITC and CD19-PE and shown in 'R2'. (iii) Dot plot of P1 gated cells, except 'R2', stained with CD20-FITC and CD3-CyQ. (B) (i) Typical flow cytometry dot plot of the forward and side scatter of PBMCs. (ii) Dot plot of P1 gated cells stained with isotype control IgG-FITC and isotype control IgG-PE and shown in 'R2' (iii). Dot plot of P1 gated cells, except 'R2', stained with isotype control IgG-FITC and isotype control CD3-CyQ. (C) (i) Typical flow cytometry dot plot of the forward and side scatter of PBMCs. (ii) Dot plot of P1 gated cells stained with CD20-FITC and CD19-PE post-RTX treatment and shown in 'R2'. (iii) Dot plot of P1 gated cells, except 'R2', stained with CD20-FITC and CD3-CyQ post-RTX incubation.

    Article Snippet: To confirm that the anti-CD20-conjugated antibodies were binding specifically to surface-expressed CD20 antigen, isolated PMBCs were washed and pre-incubated with 5 μg/mL RTX (Hoffman-La Roche, Basel, Switzerland) on ice for 15 minutes and washed again before beginning the staining procedures for flow cytometry analysis (Accuri flow cytometer; BD Biosciences, San Jose, CA, USA).

    Techniques: Selection, Flow Cytometry, Cytometry, Staining, Incubation

    Effects of PD098,059 (25 μ M) and Bay11-7082 (50 μ M) on ChAT protein expression in 16-HBE cells. Cells were preincubated for 1 h with PD098,059 (25 μ M) or Bay11-7082 (50 μ M) and then stimulated with rhIL-17A 20 ng/mL for 24 h to evaluate ChAT protein expression (a) by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI) of three separate experiments. Representative (b) flow cytometry analysis and western blot (c) are shown. Bars represent mean ± SD of arbitrary densitometric units (ADU). Representative western blot is shown. Statistical analysis was performed by ANOVA test followed by Fisher's PLSD multiple comparison test. The black curve represents the anti-IgG isotype negative control antibody.

    Journal: Mediators of Inflammation

    Article Title: Autocrine Acetylcholine, Induced by IL-17A via NFκB and ERK1/2 Pathway Activation, Promotes MUC5AC and IL-8 Synthesis in Bronchial Epithelial Cells

    doi: 10.1155/2016/9063842

    Figure Lengend Snippet: Effects of PD098,059 (25 μ M) and Bay11-7082 (50 μ M) on ChAT protein expression in 16-HBE cells. Cells were preincubated for 1 h with PD098,059 (25 μ M) or Bay11-7082 (50 μ M) and then stimulated with rhIL-17A 20 ng/mL for 24 h to evaluate ChAT protein expression (a) by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI) of three separate experiments. Representative (b) flow cytometry analysis and western blot (c) are shown. Bars represent mean ± SD of arbitrary densitometric units (ADU). Representative western blot is shown. Statistical analysis was performed by ANOVA test followed by Fisher's PLSD multiple comparison test. The black curve represents the anti-IgG isotype negative control antibody.

    Article Snippet: The cells were washed twice and then incubated with FITC-conjugated rabbit polyclonal anti-mouse IgG F(ab′)2 (Dako, Glostrup, Denmark) for 30 min at 4°C and then analyzed by flow cytometry analyses (FACSCalibur flow cytometer, Becton Dickinson, Mountain View, CA, USA) supported by CellQuest acquisition and data analysis software (Becton Dickinson, Mountain View, CA, USA).

    Techniques: Expressing, Flow Cytometry, Cytometry, Fluorescence, Western Blot, Negative Control

    Effects of PD098,059 (25 μ M), Bay11-7082 (50 μ M), and Tiotropium (100 nM) on ACh in 16-HBE cells. Cells were preincubated for 1 h with PD098,059 (25 μ M) or Bay11-7082 (50 μ M) and then stimulated with rhIL-17A 20 ng/mL for 24 h to evaluate (a) ACh binding by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI). (b) Representative flow cytometry is shown. (c) ACh production, expressed as pmoli/ μ g protein. Bars represent mean ± SD of three different experiments. Then, 16-HBE cells were preincubated for 1 h with Tiotropium (100 nM) and then stimulated with rhIL-17A 20 ng/mL for 24 h to evaluate (d) ACh binding by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI). (e) Representative flow cytometry is shown. Statistical analysis was performed by ANOVA test followed by Fisher's PLSD multiple comparison test. The black curve represents the anti-IgG isotype negative control antibody.

    Journal: Mediators of Inflammation

    Article Title: Autocrine Acetylcholine, Induced by IL-17A via NFκB and ERK1/2 Pathway Activation, Promotes MUC5AC and IL-8 Synthesis in Bronchial Epithelial Cells

    doi: 10.1155/2016/9063842

    Figure Lengend Snippet: Effects of PD098,059 (25 μ M), Bay11-7082 (50 μ M), and Tiotropium (100 nM) on ACh in 16-HBE cells. Cells were preincubated for 1 h with PD098,059 (25 μ M) or Bay11-7082 (50 μ M) and then stimulated with rhIL-17A 20 ng/mL for 24 h to evaluate (a) ACh binding by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI). (b) Representative flow cytometry is shown. (c) ACh production, expressed as pmoli/ μ g protein. Bars represent mean ± SD of three different experiments. Then, 16-HBE cells were preincubated for 1 h with Tiotropium (100 nM) and then stimulated with rhIL-17A 20 ng/mL for 24 h to evaluate (d) ACh binding by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI). (e) Representative flow cytometry is shown. Statistical analysis was performed by ANOVA test followed by Fisher's PLSD multiple comparison test. The black curve represents the anti-IgG isotype negative control antibody.

    Article Snippet: The cells were washed twice and then incubated with FITC-conjugated rabbit polyclonal anti-mouse IgG F(ab′)2 (Dako, Glostrup, Denmark) for 30 min at 4°C and then analyzed by flow cytometry analyses (FACSCalibur flow cytometer, Becton Dickinson, Mountain View, CA, USA) supported by CellQuest acquisition and data analysis software (Becton Dickinson, Mountain View, CA, USA).

    Techniques: Binding Assay, Flow Cytometry, Cytometry, Fluorescence, Negative Control

    rhIL-17A increased endogenous ACh binding and production in 16-HBE cells. Cells were stimulated with rhIL-17A (0–50 ng/mL) for 24 h to evaluate (a) ACh binding by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI) of three separate experiments. (b) Representative flow cytometry analysis is shown. (c) ACh production expressed as pmoli/ μ g protein. Bars represent mean ± SD of three different experiments. Statistical analysis was performed by ANOVA test followed by Fisher's PLSD multiple comparison test. The black curve represents the anti-IgG isotype negative control antibody.

    Journal: Mediators of Inflammation

    Article Title: Autocrine Acetylcholine, Induced by IL-17A via NFκB and ERK1/2 Pathway Activation, Promotes MUC5AC and IL-8 Synthesis in Bronchial Epithelial Cells

    doi: 10.1155/2016/9063842

    Figure Lengend Snippet: rhIL-17A increased endogenous ACh binding and production in 16-HBE cells. Cells were stimulated with rhIL-17A (0–50 ng/mL) for 24 h to evaluate (a) ACh binding by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI) of three separate experiments. (b) Representative flow cytometry analysis is shown. (c) ACh production expressed as pmoli/ μ g protein. Bars represent mean ± SD of three different experiments. Statistical analysis was performed by ANOVA test followed by Fisher's PLSD multiple comparison test. The black curve represents the anti-IgG isotype negative control antibody.

    Article Snippet: The cells were washed twice and then incubated with FITC-conjugated rabbit polyclonal anti-mouse IgG F(ab′)2 (Dako, Glostrup, Denmark) for 30 min at 4°C and then analyzed by flow cytometry analyses (FACSCalibur flow cytometer, Becton Dickinson, Mountain View, CA, USA) supported by CellQuest acquisition and data analysis software (Becton Dickinson, Mountain View, CA, USA).

    Techniques: Binding Assay, Flow Cytometry, Cytometry, Fluorescence, Negative Control

    Effect of Tiotropium on ChAT, Muc5AC, and IL-8 in N-HBE cells. Cells were incubated for 1 h with Tiotropium (100 nM) before addition of rhIL-17A 20 ng/mL for 24 h to evaluate (a) ChAT protein expression by western blot. Bars represent mean ± SD of arbitrary densitometric units (ADU). Representative western blot analysis of ChAT protein and β -actin is shown. (b) Muc5AC protein expression by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI) of three separate experiments and were plotted as fold change compared to untreated cells. (c) Muc5AC protein expression by western blot. Bars represent mean ± SD of arbitrary densitometric units (ADU) of three different experiments. Representative western blot analysis of Muc5AC protein is shown. (d) IL-8 expression by western blot. Bars represent mean ± SD of arbitrary densitometric units (ADU). Representative western blot analyses of IL-8 protein and β -actin are shown. Statistical analysis was performed by ANOVA followed by Fisher's PLSD multiple comparison test. p

    Journal: Mediators of Inflammation

    Article Title: Autocrine Acetylcholine, Induced by IL-17A via NFκB and ERK1/2 Pathway Activation, Promotes MUC5AC and IL-8 Synthesis in Bronchial Epithelial Cells

    doi: 10.1155/2016/9063842

    Figure Lengend Snippet: Effect of Tiotropium on ChAT, Muc5AC, and IL-8 in N-HBE cells. Cells were incubated for 1 h with Tiotropium (100 nM) before addition of rhIL-17A 20 ng/mL for 24 h to evaluate (a) ChAT protein expression by western blot. Bars represent mean ± SD of arbitrary densitometric units (ADU). Representative western blot analysis of ChAT protein and β -actin is shown. (b) Muc5AC protein expression by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI) of three separate experiments and were plotted as fold change compared to untreated cells. (c) Muc5AC protein expression by western blot. Bars represent mean ± SD of arbitrary densitometric units (ADU) of three different experiments. Representative western blot analysis of Muc5AC protein is shown. (d) IL-8 expression by western blot. Bars represent mean ± SD of arbitrary densitometric units (ADU). Representative western blot analyses of IL-8 protein and β -actin are shown. Statistical analysis was performed by ANOVA followed by Fisher's PLSD multiple comparison test. p

    Article Snippet: The cells were washed twice and then incubated with FITC-conjugated rabbit polyclonal anti-mouse IgG F(ab′)2 (Dako, Glostrup, Denmark) for 30 min at 4°C and then analyzed by flow cytometry analyses (FACSCalibur flow cytometer, Becton Dickinson, Mountain View, CA, USA) supported by CellQuest acquisition and data analysis software (Becton Dickinson, Mountain View, CA, USA).

    Techniques: Incubation, Expressing, Western Blot, Flow Cytometry, Cytometry, Fluorescence

    Effect of Hemicholinium-3 (HCh-3) or Tiotropium on Muc5AC production in 16-HBE cells. Cells were incubated for 1 h with HCh-3 (50 μ M) or Tiotropium (100 nM) before addition of rhIL-17A 20 ng/mL for 24 h to evaluate ((a)–(d)) Muc5AC protein expression by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI) of three separate experiments. Data were plotted as fold change compared to untreated cells. Representative flow cytometry of Muc5AC is shown. ((e)-(f)) Muc5AC protein expression by western blot. Bars represent mean ± SD of arbitrary densitometric units (ADU) of three different experiments. Representative western blot analysis of Muc5AC protein is shown. Statistical analysis was performed by ANOVA followed by Fisher's PLSD multiple comparison test. The black curve represents the anti-IgG isotype negative control antibody.

    Journal: Mediators of Inflammation

    Article Title: Autocrine Acetylcholine, Induced by IL-17A via NFκB and ERK1/2 Pathway Activation, Promotes MUC5AC and IL-8 Synthesis in Bronchial Epithelial Cells

    doi: 10.1155/2016/9063842

    Figure Lengend Snippet: Effect of Hemicholinium-3 (HCh-3) or Tiotropium on Muc5AC production in 16-HBE cells. Cells were incubated for 1 h with HCh-3 (50 μ M) or Tiotropium (100 nM) before addition of rhIL-17A 20 ng/mL for 24 h to evaluate ((a)–(d)) Muc5AC protein expression by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI) of three separate experiments. Data were plotted as fold change compared to untreated cells. Representative flow cytometry of Muc5AC is shown. ((e)-(f)) Muc5AC protein expression by western blot. Bars represent mean ± SD of arbitrary densitometric units (ADU) of three different experiments. Representative western blot analysis of Muc5AC protein is shown. Statistical analysis was performed by ANOVA followed by Fisher's PLSD multiple comparison test. The black curve represents the anti-IgG isotype negative control antibody.

    Article Snippet: The cells were washed twice and then incubated with FITC-conjugated rabbit polyclonal anti-mouse IgG F(ab′)2 (Dako, Glostrup, Denmark) for 30 min at 4°C and then analyzed by flow cytometry analyses (FACSCalibur flow cytometer, Becton Dickinson, Mountain View, CA, USA) supported by CellQuest acquisition and data analysis software (Becton Dickinson, Mountain View, CA, USA).

    Techniques: Incubation, Expressing, Flow Cytometry, Cytometry, Fluorescence, Western Blot, Negative Control

    Silencing of ChAT mRNA reduced Muc5AC expression and IL-8 release in 16-HBE cells stimulated with rhIL-17A 20 ng/mL for 24 h. Cells were stimulated and analyzed to evaluate (a) Muc5AC expression in unsilenced cells and in cells transfected with siRNA for ChAT or scrambled siRNA by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI) of three separate experiments. (b) Representative flow cytometry of Muc5AC is shown. (c) IL-8 release (pg/mL, by ELISA) in unsilenced cells and in cells transfected with siRNA for ChAT or scrambled siRNA. The values shown are the mean ± SD for three separated experiments. (d) Knockdown efficiency (%) of ChAT mRNA on ChAT protein expression. Bars represent mean ± SD of three separate experiments. Statistical analysis was performed by ANOVA test followed by Fisher's PLSD multiple comparison test.

    Journal: Mediators of Inflammation

    Article Title: Autocrine Acetylcholine, Induced by IL-17A via NFκB and ERK1/2 Pathway Activation, Promotes MUC5AC and IL-8 Synthesis in Bronchial Epithelial Cells

    doi: 10.1155/2016/9063842

    Figure Lengend Snippet: Silencing of ChAT mRNA reduced Muc5AC expression and IL-8 release in 16-HBE cells stimulated with rhIL-17A 20 ng/mL for 24 h. Cells were stimulated and analyzed to evaluate (a) Muc5AC expression in unsilenced cells and in cells transfected with siRNA for ChAT or scrambled siRNA by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI) of three separate experiments. (b) Representative flow cytometry of Muc5AC is shown. (c) IL-8 release (pg/mL, by ELISA) in unsilenced cells and in cells transfected with siRNA for ChAT or scrambled siRNA. The values shown are the mean ± SD for three separated experiments. (d) Knockdown efficiency (%) of ChAT mRNA on ChAT protein expression. Bars represent mean ± SD of three separate experiments. Statistical analysis was performed by ANOVA test followed by Fisher's PLSD multiple comparison test.

    Article Snippet: The cells were washed twice and then incubated with FITC-conjugated rabbit polyclonal anti-mouse IgG F(ab′)2 (Dako, Glostrup, Denmark) for 30 min at 4°C and then analyzed by flow cytometry analyses (FACSCalibur flow cytometer, Becton Dickinson, Mountain View, CA, USA) supported by CellQuest acquisition and data analysis software (Becton Dickinson, Mountain View, CA, USA).

    Techniques: Expressing, Transfection, Flow Cytometry, Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay

    rhIL-17A increased ChAT protein expression and mRNA in 16-HBE cells. Cells were stimulated with rhIL-17A (0–50 ng/mL) for 24 h to evaluate ChAT protein expression (a) by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI) of three separate experiments. Representative (b) flow cytometry analysis and western blot (c) are shown. Bars represent mean ± SD of arbitrary densitometric units (ADU). Representative western blot analysis of ChAT protein and β -actin is shown. (d) Cells were stimulated with rhIL-17A (0–20 ng/mL) for 24 h to evaluate ChAT mRNA levels by RT-PCR. Bars represent mean ± SD of arbitrary units of three separate experiments and were plotted as fold change compared to untreated cells. Statistical analysis was performed by ANOVA test followed by Fisher's PLSD multiple comparison test or Student's t -test. The black curve represents the anti-IgG isotype negative control antibody.

    Journal: Mediators of Inflammation

    Article Title: Autocrine Acetylcholine, Induced by IL-17A via NFκB and ERK1/2 Pathway Activation, Promotes MUC5AC and IL-8 Synthesis in Bronchial Epithelial Cells

    doi: 10.1155/2016/9063842

    Figure Lengend Snippet: rhIL-17A increased ChAT protein expression and mRNA in 16-HBE cells. Cells were stimulated with rhIL-17A (0–50 ng/mL) for 24 h to evaluate ChAT protein expression (a) by flow cytometry. Bars represent mean ± SD of fluorescence mean intensity (FMI) of three separate experiments. Representative (b) flow cytometry analysis and western blot (c) are shown. Bars represent mean ± SD of arbitrary densitometric units (ADU). Representative western blot analysis of ChAT protein and β -actin is shown. (d) Cells were stimulated with rhIL-17A (0–20 ng/mL) for 24 h to evaluate ChAT mRNA levels by RT-PCR. Bars represent mean ± SD of arbitrary units of three separate experiments and were plotted as fold change compared to untreated cells. Statistical analysis was performed by ANOVA test followed by Fisher's PLSD multiple comparison test or Student's t -test. The black curve represents the anti-IgG isotype negative control antibody.

    Article Snippet: The cells were washed twice and then incubated with FITC-conjugated rabbit polyclonal anti-mouse IgG F(ab′)2 (Dako, Glostrup, Denmark) for 30 min at 4°C and then analyzed by flow cytometry analyses (FACSCalibur flow cytometer, Becton Dickinson, Mountain View, CA, USA) supported by CellQuest acquisition and data analysis software (Becton Dickinson, Mountain View, CA, USA).

    Techniques: Expressing, Flow Cytometry, Cytometry, Fluorescence, Western Blot, Reverse Transcription Polymerase Chain Reaction, Negative Control

    Dasatinib blocks antigen-induced TCR downregulation and tetramer internalization from the cell surface. A. Mel13 CTL were pre-treated with PBS ± 50 nM dasatinib and exposed to C1R-A2 B cells previously pulsed with 10 -6 M ELAGIGILTV peptide or medium alone for 4 h at 37 °C. Cells were subsequently stained with anti-TCR-FITC (clone BMA 031; Serotec) and anti-CD8-APC (clone RPA-T8; BD Pharmingen) mAbs for 30 min on ice, washed twice and resuspended in PBS. Data were acquired on a FACSCalibur flow cytometer (BD) and analyzed using FlowJo software. B. 10 5 ILA1 CTL were pre-treated with PBS (i ii) or PBS + 50 nM dasatinib (iii iv) for 30 min at 37 °C, then stained with 20μg/ml HLA A2/ILAKFLHWL-Alexa488 tetramer for 15 min at 37 °C. Microscopy was performed as described in the Materials and methods.

    Journal: Journal of Immunological Methods

    Article Title: Protein kinase inhibitors substantially improve the physical detection of T-cells with peptide-MHC tetramers

    doi: 10.1016/j.jim.2008.09.014

    Figure Lengend Snippet: Dasatinib blocks antigen-induced TCR downregulation and tetramer internalization from the cell surface. A. Mel13 CTL were pre-treated with PBS ± 50 nM dasatinib and exposed to C1R-A2 B cells previously pulsed with 10 -6 M ELAGIGILTV peptide or medium alone for 4 h at 37 °C. Cells were subsequently stained with anti-TCR-FITC (clone BMA 031; Serotec) and anti-CD8-APC (clone RPA-T8; BD Pharmingen) mAbs for 30 min on ice, washed twice and resuspended in PBS. Data were acquired on a FACSCalibur flow cytometer (BD) and analyzed using FlowJo software. B. 10 5 ILA1 CTL were pre-treated with PBS (i ii) or PBS + 50 nM dasatinib (iii iv) for 30 min at 37 °C, then stained with 20μg/ml HLA A2/ILAKFLHWL-Alexa488 tetramer for 15 min at 37 °C. Microscopy was performed as described in the Materials and methods.

    Article Snippet: The cells were evaluated using a FACSCalibur flow cytometer (BD Biosciences).

    Techniques: CTL Assay, Staining, Recombinase Polymerase Amplification, Flow Cytometry, Cytometry, Software, Microscopy

    Dasatinib results in a time dependent increase in TCR and CD8 expression levels at the CTL cell surface. The ILA1 CTL clone was treated with PBS ± 50 nM dasatinib at 37 °C and 10 5 CTL were removed from the medium at 0, 10, 30, 60, 180 and 250 min. CTL were subsequently stained with anti-CD8 FITC (clone SK1; BD, Pharmingen; left panel) or anti-TCR FITC (clone BMA 031; Serotec; right panel) for 30 min on ice, washed twice and resuspended in PBS. Data were acquired on a FACSCalibur flow cytometer (BD) and analyzed using FlowJo software.

    Journal: Journal of Immunological Methods

    Article Title: Protein kinase inhibitors substantially improve the physical detection of T-cells with peptide-MHC tetramers

    doi: 10.1016/j.jim.2008.09.014

    Figure Lengend Snippet: Dasatinib results in a time dependent increase in TCR and CD8 expression levels at the CTL cell surface. The ILA1 CTL clone was treated with PBS ± 50 nM dasatinib at 37 °C and 10 5 CTL were removed from the medium at 0, 10, 30, 60, 180 and 250 min. CTL were subsequently stained with anti-CD8 FITC (clone SK1; BD, Pharmingen; left panel) or anti-TCR FITC (clone BMA 031; Serotec; right panel) for 30 min on ice, washed twice and resuspended in PBS. Data were acquired on a FACSCalibur flow cytometer (BD) and analyzed using FlowJo software.

    Article Snippet: The cells were evaluated using a FACSCalibur flow cytometer (BD Biosciences).

    Techniques: Expressing, CTL Assay, Staining, Flow Cytometry, Cytometry, Software

    Beneficial effects of dasatinib are not CD8-mediated. A. Melc5 CTL were pre- treated with PBS ± 50 nM dasatinib for 30 min at 37 °C, then stained with HLA A2 DT227/8KA cognate tetramer for 20 min at 37 °C. After washing twice, data were acquired on a FACSCalibur flow cytometer (BD) and analyzed using FlowJo software. B. Staining of HLA A2-restricted CTL lines expanded from PBMC by one round of stimulation with the Melan-A/Mart-1 26-35 peptide (ELAGIGILTV). Lines were stained with either wild type or CD8 null cognate tetramer ± pre-treatment with 50 nM dasatinib for 30 min at 37 °C.

    Journal: Journal of Immunological Methods

    Article Title: Protein kinase inhibitors substantially improve the physical detection of T-cells with peptide-MHC tetramers

    doi: 10.1016/j.jim.2008.09.014

    Figure Lengend Snippet: Beneficial effects of dasatinib are not CD8-mediated. A. Melc5 CTL were pre- treated with PBS ± 50 nM dasatinib for 30 min at 37 °C, then stained with HLA A2 DT227/8KA cognate tetramer for 20 min at 37 °C. After washing twice, data were acquired on a FACSCalibur flow cytometer (BD) and analyzed using FlowJo software. B. Staining of HLA A2-restricted CTL lines expanded from PBMC by one round of stimulation with the Melan-A/Mart-1 26-35 peptide (ELAGIGILTV). Lines were stained with either wild type or CD8 null cognate tetramer ± pre-treatment with 50 nM dasatinib for 30 min at 37 °C.

    Article Snippet: The cells were evaluated using a FACSCalibur flow cytometer (BD Biosciences).

    Techniques: CTL Assay, Staining, Flow Cytometry, Cytometry, Software

    Dasatinib substantially improves pMHC tetramer staining intensity. A. 10 5 ILA1 CTL were re-suspended in 40μl of PBS ± 50 nM dasatinib or Lck inhibitor II (Calbiochem), then incubated at 37 °C for 30 min. Cells were then stained with cognate HLA A2/ILAKFLHWL-PE tetramer at a final concentration of 10µg/ml for 20 min at 37 °C, washed twice in PBS and analyzed on a FACSCalibur (BD) flow cytometer. A > 10-fold increase in median fluorescence intensity (MFI) was observed after treatment with 50 nM dasatinib (blue) or LcK inhibitor II (red) compared to staining without PKI pre-treatment (green line). B. 10 5 ILA1 CTL were treated with various concentrations of dasatinib for 30 min at 37 °C, then stained with either HLA A2/ILAKFLHWL tetramer or the non-cognate HLA A2/ELAGIGILTV tetramer for 20 min at 37 °C before washing with PBS. C. 10 5 ILA1 CTL were resuspended in 40μl of PBS ± the indicated concentration of dasatinib and incubated for 60 min at 37 °C. Cells were then stained with cognate HLA A2/ILAKFLHWL-PE tetramer at a final concentration of 10 μg/ml for 20 min at 37 °C and washed twice in PBS prior to flow cytometric analysis. D. As (A), but ILA1 CTL were incubated with 50 nM dasatinib for various times prior to staining with pMHCI tetramer. For this experiment the drug was washed off prior to staining. E. As (A), but tetramer concentration was varied to stain CTL pre-treated ± 50 nM dasatinib for 30 min. F. 10 5 Mel13 CTL were stained with various concentrations of HLA A2/ELAGIGILTV tetramer following incubation ± 50 nM dasatinib for 30 min. G. 5x10 5 splenocytes from an F5 TCR transgenic Rag + mouse were resuspended in PBS ± 50 nM dasatinib and incubated for 30 min at 37 °C. Cells were subsequently stained with H2-D b /ASNENMDAM-PE tetramer for 20 min at 37 °C followed by anti-CD8 Cy5.5 for 30 min on ice prior to two washes in PBS and analysis by flow cytometry. H. 10 5 cells of the HLA DR⁎0101-restricted, influenza virus A HA 307-319 PKYVKQNTLKLAT-specific CD4 + clone C6 were incubated with PBS ± 50 nM dasatinib for 30 min at 37 °C, then stained with cognate PE-conjugated tetramer for 20 min at 37 °C. Samples were washed with PBS before flow cytometric analysis. Irrelevant tetramer was used as a negative control in all cases.

    Journal: Journal of Immunological Methods

    Article Title: Protein kinase inhibitors substantially improve the physical detection of T-cells with peptide-MHC tetramers

    doi: 10.1016/j.jim.2008.09.014

    Figure Lengend Snippet: Dasatinib substantially improves pMHC tetramer staining intensity. A. 10 5 ILA1 CTL were re-suspended in 40μl of PBS ± 50 nM dasatinib or Lck inhibitor II (Calbiochem), then incubated at 37 °C for 30 min. Cells were then stained with cognate HLA A2/ILAKFLHWL-PE tetramer at a final concentration of 10µg/ml for 20 min at 37 °C, washed twice in PBS and analyzed on a FACSCalibur (BD) flow cytometer. A > 10-fold increase in median fluorescence intensity (MFI) was observed after treatment with 50 nM dasatinib (blue) or LcK inhibitor II (red) compared to staining without PKI pre-treatment (green line). B. 10 5 ILA1 CTL were treated with various concentrations of dasatinib for 30 min at 37 °C, then stained with either HLA A2/ILAKFLHWL tetramer or the non-cognate HLA A2/ELAGIGILTV tetramer for 20 min at 37 °C before washing with PBS. C. 10 5 ILA1 CTL were resuspended in 40μl of PBS ± the indicated concentration of dasatinib and incubated for 60 min at 37 °C. Cells were then stained with cognate HLA A2/ILAKFLHWL-PE tetramer at a final concentration of 10 μg/ml for 20 min at 37 °C and washed twice in PBS prior to flow cytometric analysis. D. As (A), but ILA1 CTL were incubated with 50 nM dasatinib for various times prior to staining with pMHCI tetramer. For this experiment the drug was washed off prior to staining. E. As (A), but tetramer concentration was varied to stain CTL pre-treated ± 50 nM dasatinib for 30 min. F. 10 5 Mel13 CTL were stained with various concentrations of HLA A2/ELAGIGILTV tetramer following incubation ± 50 nM dasatinib for 30 min. G. 5x10 5 splenocytes from an F5 TCR transgenic Rag + mouse were resuspended in PBS ± 50 nM dasatinib and incubated for 30 min at 37 °C. Cells were subsequently stained with H2-D b /ASNENMDAM-PE tetramer for 20 min at 37 °C followed by anti-CD8 Cy5.5 for 30 min on ice prior to two washes in PBS and analysis by flow cytometry. H. 10 5 cells of the HLA DR⁎0101-restricted, influenza virus A HA 307-319 PKYVKQNTLKLAT-specific CD4 + clone C6 were incubated with PBS ± 50 nM dasatinib for 30 min at 37 °C, then stained with cognate PE-conjugated tetramer for 20 min at 37 °C. Samples were washed with PBS before flow cytometric analysis. Irrelevant tetramer was used as a negative control in all cases.

    Article Snippet: The cells were evaluated using a FACSCalibur flow cytometer (BD Biosciences).

    Techniques: Staining, CTL Assay, Incubation, Concentration Assay, Flow Cytometry, Cytometry, Fluorescence, Transgenic Assay, Hemagglutination Assay, Negative Control

    Dasatinib treatment preferentially increases the ability of pMHCI tetramers to stain T-cells bearing low affinity TCRs. A. 10 5 ILA1 CTL were stained with 10μg/ml PE-conjugated HLA A2 tetramer folded around the 8E, 5Y, 4L, index (ILAKFLHWL), 3G8T or 3G peptides for 20 min at 37 °C following incubation ± 50 nM dasatinib for 30 min at 37 °C. For all samples, data were acquired with a FACSCalibur flow cytometer (BD) and analyzed using FlowJo software. Irrelevant tetramer was used as a negative control. B. The MFI of tetramer staining for all of the variants in the presence and absence of dasatinib displayed in (A) are plotted against the monomeric affinity of TCR/pMHCI interactions previously measured for each of these variants expressed as the dissociation constant (K D ) ( Table 1 ). Curves were fitted as described in the Materials and methods.

    Journal: Journal of Immunological Methods

    Article Title: Protein kinase inhibitors substantially improve the physical detection of T-cells with peptide-MHC tetramers

    doi: 10.1016/j.jim.2008.09.014

    Figure Lengend Snippet: Dasatinib treatment preferentially increases the ability of pMHCI tetramers to stain T-cells bearing low affinity TCRs. A. 10 5 ILA1 CTL were stained with 10μg/ml PE-conjugated HLA A2 tetramer folded around the 8E, 5Y, 4L, index (ILAKFLHWL), 3G8T or 3G peptides for 20 min at 37 °C following incubation ± 50 nM dasatinib for 30 min at 37 °C. For all samples, data were acquired with a FACSCalibur flow cytometer (BD) and analyzed using FlowJo software. Irrelevant tetramer was used as a negative control. B. The MFI of tetramer staining for all of the variants in the presence and absence of dasatinib displayed in (A) are plotted against the monomeric affinity of TCR/pMHCI interactions previously measured for each of these variants expressed as the dissociation constant (K D ) ( Table 1 ). Curves were fitted as described in the Materials and methods.

    Article Snippet: The cells were evaluated using a FACSCalibur flow cytometer (BD Biosciences).

    Techniques: Staining, CTL Assay, Incubation, Flow Cytometry, Cytometry, Software, Negative Control

    Effect of different TLR2 complex agonists on the proliferation of GFP + Treg. FACS-sorted GFP + Treg (5×10 4 ) were cultured w ith plate-bound anti-CD3 (5µg/ml) and IL-2 for 3 days in the presence or absence of: A , TLR2/TLR2 agonist, LTA-SA;

    Journal:

    Article Title: Engagement of TLR2 Does not Reverse the Suppressor Function of Mouse Regulatory T Cells, but Promotes Their Survival

    doi: 10.4049/jimmunol.0901465

    Figure Lengend Snippet: Effect of different TLR2 complex agonists on the proliferation of GFP + Treg. FACS-sorted GFP + Treg (5×10 4 ) were cultured w ith plate-bound anti-CD3 (5µg/ml) and IL-2 for 3 days in the presence or absence of: A , TLR2/TLR2 agonist, LTA-SA;

    Article Snippet: The enriched CD4+ fraction were then further separated into conventional T or Treg populations by FACS sorting for the CD4+ CD25− , CD4+ GFP− or CD4+ GFP+ fractions using either the FACs Vantage DiVa or FACs Aria flow cytometer (BD Biosciences, San Jose, CA).

    Techniques: FACS, Cell Culture

    Effect of TLR agonists on the proliferation of GFP + Treg and GFP − T cells in vitro . A and B , FACS-sorted GFP + Treg (5×10 4 ) were cultured for 3 days with T-depleted spleen cells and soluble anti-CD3 (1µg/ml) (A) or with plate-bound

    Journal:

    Article Title: Engagement of TLR2 Does not Reverse the Suppressor Function of Mouse Regulatory T Cells, but Promotes Their Survival

    doi: 10.4049/jimmunol.0901465

    Figure Lengend Snippet: Effect of TLR agonists on the proliferation of GFP + Treg and GFP − T cells in vitro . A and B , FACS-sorted GFP + Treg (5×10 4 ) were cultured for 3 days with T-depleted spleen cells and soluble anti-CD3 (1µg/ml) (A) or with plate-bound

    Article Snippet: The enriched CD4+ fraction were then further separated into conventional T or Treg populations by FACS sorting for the CD4+ CD25− , CD4+ GFP− or CD4+ GFP+ fractions using either the FACs Vantage DiVa or FACs Aria flow cytometer (BD Biosciences, San Jose, CA).

    Techniques: In Vitro, FACS, Cell Culture

    Pam3CSK4 augments Treg survival and induces Bcl-x L but not Bcl-2 expression. A , FACS-sorted GFP + Treg were cultured with plate-bound anti-CD3 (0.4µg/ml) and IL-2 for 7 days in the absence (left panel) or presence (right panel) of Pam3CSK4. The

    Journal:

    Article Title: Engagement of TLR2 Does not Reverse the Suppressor Function of Mouse Regulatory T Cells, but Promotes Their Survival

    doi: 10.4049/jimmunol.0901465

    Figure Lengend Snippet: Pam3CSK4 augments Treg survival and induces Bcl-x L but not Bcl-2 expression. A , FACS-sorted GFP + Treg were cultured with plate-bound anti-CD3 (0.4µg/ml) and IL-2 for 7 days in the absence (left panel) or presence (right panel) of Pam3CSK4. The

    Article Snippet: The enriched CD4+ fraction were then further separated into conventional T or Treg populations by FACS sorting for the CD4+ CD25− , CD4+ GFP− or CD4+ GFP+ fractions using either the FACs Vantage DiVa or FACs Aria flow cytometer (BD Biosciences, San Jose, CA).

    Techniques: Expressing, FACS, Cell Culture

    Pam3CSK4 stimulation does not alter Foxp3 expression or Treg suppressive activity. A , FACS-sorted GFP + Treg were stimulated with plate-bound anti-CD3 and IL-2 for 3 days in the presence or absence of Pam3CSK4. Flow cytometric analysis of GFP expression

    Journal:

    Article Title: Engagement of TLR2 Does not Reverse the Suppressor Function of Mouse Regulatory T Cells, but Promotes Their Survival

    doi: 10.4049/jimmunol.0901465

    Figure Lengend Snippet: Pam3CSK4 stimulation does not alter Foxp3 expression or Treg suppressive activity. A , FACS-sorted GFP + Treg were stimulated with plate-bound anti-CD3 and IL-2 for 3 days in the presence or absence of Pam3CSK4. Flow cytometric analysis of GFP expression

    Article Snippet: The enriched CD4+ fraction were then further separated into conventional T or Treg populations by FACS sorting for the CD4+ CD25− , CD4+ GFP− or CD4+ GFP+ fractions using either the FACs Vantage DiVa or FACs Aria flow cytometer (BD Biosciences, San Jose, CA).

    Techniques: Expressing, Activity Assay, FACS, Flow Cytometry

    Expression of TLR2 mRNA in GFP + Treg and GFP − T cells. A , FACS-sorted GFP − T cells or GFP + Treg (5×10 4 ) were either un-stimulated or activated by plate-bound anti-CD3 in the absence or presence of Pam3CSK4 for 16h. IL-2 was added

    Journal:

    Article Title: Engagement of TLR2 Does not Reverse the Suppressor Function of Mouse Regulatory T Cells, but Promotes Their Survival

    doi: 10.4049/jimmunol.0901465

    Figure Lengend Snippet: Expression of TLR2 mRNA in GFP + Treg and GFP − T cells. A , FACS-sorted GFP − T cells or GFP + Treg (5×10 4 ) were either un-stimulated or activated by plate-bound anti-CD3 in the absence or presence of Pam3CSK4 for 16h. IL-2 was added

    Article Snippet: The enriched CD4+ fraction were then further separated into conventional T or Treg populations by FACS sorting for the CD4+ CD25− , CD4+ GFP− or CD4+ GFP+ fractions using either the FACs Vantage DiVa or FACs Aria flow cytometer (BD Biosciences, San Jose, CA).

    Techniques: Expressing, FACS

    ZIP6 enrichment in non-adherent cells ( A ) CHO or MCF-7 cells transfected with lipid control, ZIP6, ZIP7 or β-galactosidase (LacZ) were imaged 16 h after transfection and cells transfected with ZIP6 had either detached clumps of live cells (CHO) or detached single round cells (MCF-7). Non-adherent MCF-7 cells grown overnight on new coverslips for 24 h (re-adhered) had more cells from ZIP6 transfected dishes. ( B ) Adherent (grey) and non-adherent (black) MCF-7 cells treated for 7 days with oestrogen (E2), EGF, TGF, TAM or FAS (Faslodex®) were collected and probed for ZIP6 conjugated with Alexa Fluor® 488 and analysed by FACS. Results are means±S.D. for three experiments and demonstrate ZIP6 enrichment in non-adherent cells. Statistical significance from the relevant control (CON) is indicated above the histogram bars as * P

    Journal: Biochemical Journal

    Article Title: A mechanism for epithelial-mesenchymal transition and anoikis resistance in breast cancer triggered by zinc channel ZIP6 and STAT3 (signal transducer and activator of transcription 3)

    doi: 10.1042/BJ20130483

    Figure Lengend Snippet: ZIP6 enrichment in non-adherent cells ( A ) CHO or MCF-7 cells transfected with lipid control, ZIP6, ZIP7 or β-galactosidase (LacZ) were imaged 16 h after transfection and cells transfected with ZIP6 had either detached clumps of live cells (CHO) or detached single round cells (MCF-7). Non-adherent MCF-7 cells grown overnight on new coverslips for 24 h (re-adhered) had more cells from ZIP6 transfected dishes. ( B ) Adherent (grey) and non-adherent (black) MCF-7 cells treated for 7 days with oestrogen (E2), EGF, TGF, TAM or FAS (Faslodex®) were collected and probed for ZIP6 conjugated with Alexa Fluor® 488 and analysed by FACS. Results are means±S.D. for three experiments and demonstrate ZIP6 enrichment in non-adherent cells. Statistical significance from the relevant control (CON) is indicated above the histogram bars as * P

    Article Snippet: A BD Biosciences FACS III flow cytometer and software was used to analyse the FACS results.

    Techniques: Transfection, FACS

    ZIP6 and ELP2 are regulated by a shared 5′ region ( A ) Annotated genomic region on human chromosome 18, including the 5′-teminal regions of SLC39A6 ( ZIP6 ) and ELP2 ( STATIP1 ). Coding regions are shown as filled boxes and UTRs of exons as open boxes. Motifs for ER (Transfac ER motif M00191) and STAT3 (Transfac STAT3 motif M00225) are denoted by red and blue vertical lines respectively. The direction of the two genes in the genomic sequence is shown by golden arrows. ( B ) Immunocytochemical analysis showing increase in ZIP6 and Snail in MCF-7 cells treated with oestrogen (E2) for 7 days (brown). The nucleus was counterstained blue. ( C ) Loss of E-cadherin (green) only in recombinant ZIP6-positive cell probed with anti-V5 antibody (red). ( D ) MCF-7 transfected cells were probed for activated GSK-3β and analysed by FACS analysis using Alexa Fluor® 488. Results are means±S.D. for three experiments. Only cells transfected with ZIP6 show inhibition of GSK-3β. CON, control.

    Journal: Biochemical Journal

    Article Title: A mechanism for epithelial-mesenchymal transition and anoikis resistance in breast cancer triggered by zinc channel ZIP6 and STAT3 (signal transducer and activator of transcription 3)

    doi: 10.1042/BJ20130483

    Figure Lengend Snippet: ZIP6 and ELP2 are regulated by a shared 5′ region ( A ) Annotated genomic region on human chromosome 18, including the 5′-teminal regions of SLC39A6 ( ZIP6 ) and ELP2 ( STATIP1 ). Coding regions are shown as filled boxes and UTRs of exons as open boxes. Motifs for ER (Transfac ER motif M00191) and STAT3 (Transfac STAT3 motif M00225) are denoted by red and blue vertical lines respectively. The direction of the two genes in the genomic sequence is shown by golden arrows. ( B ) Immunocytochemical analysis showing increase in ZIP6 and Snail in MCF-7 cells treated with oestrogen (E2) for 7 days (brown). The nucleus was counterstained blue. ( C ) Loss of E-cadherin (green) only in recombinant ZIP6-positive cell probed with anti-V5 antibody (red). ( D ) MCF-7 transfected cells were probed for activated GSK-3β and analysed by FACS analysis using Alexa Fluor® 488. Results are means±S.D. for three experiments. Only cells transfected with ZIP6 show inhibition of GSK-3β. CON, control.

    Article Snippet: A BD Biosciences FACS III flow cytometer and software was used to analyse the FACS results.

    Techniques: Sequencing, Recombinant, Transfection, FACS, Inhibition

    ZIP6-positive cells have increased G 2 /M-phase and increased migration ( A ) CHO and MCF-7 cells transfected with control (CON), LacZ, ZIP7 or ZIP6 were harvested as adherent or non-adherent populations and tested for cell-cycle stage by FACS analysis. Both adherent and non-adherent ZIP6-positive cells had increased cells in G 2 /M-phase suggesting anoikis resistance. Values are means±S.D. for three experiments. Statistical significance of *** P

    Journal: Biochemical Journal

    Article Title: A mechanism for epithelial-mesenchymal transition and anoikis resistance in breast cancer triggered by zinc channel ZIP6 and STAT3 (signal transducer and activator of transcription 3)

    doi: 10.1042/BJ20130483

    Figure Lengend Snippet: ZIP6-positive cells have increased G 2 /M-phase and increased migration ( A ) CHO and MCF-7 cells transfected with control (CON), LacZ, ZIP7 or ZIP6 were harvested as adherent or non-adherent populations and tested for cell-cycle stage by FACS analysis. Both adherent and non-adherent ZIP6-positive cells had increased cells in G 2 /M-phase suggesting anoikis resistance. Values are means±S.D. for three experiments. Statistical significance of *** P

    Article Snippet: A BD Biosciences FACS III flow cytometer and software was used to analyse the FACS results.

    Techniques: Migration, Transfection, FACS

    ZIP6 expression requires STAT3 Fluorescent microscopy ( A ) or FACS analysis ( B ) of MCF-7 cells treated with EGF and/or a STAT3 inhibitor (STI) for 24 h and probed with ZIP6-Y antibody. The observed increase in ZIP6 by EGF treatment is prevented by the STAT3 inhibitor. FACS results show mean fluorescence±S.D. for three experiments. Statistical significance of EGF compared with control of P

    Journal: Biochemical Journal

    Article Title: A mechanism for epithelial-mesenchymal transition and anoikis resistance in breast cancer triggered by zinc channel ZIP6 and STAT3 (signal transducer and activator of transcription 3)

    doi: 10.1042/BJ20130483

    Figure Lengend Snippet: ZIP6 expression requires STAT3 Fluorescent microscopy ( A ) or FACS analysis ( B ) of MCF-7 cells treated with EGF and/or a STAT3 inhibitor (STI) for 24 h and probed with ZIP6-Y antibody. The observed increase in ZIP6 by EGF treatment is prevented by the STAT3 inhibitor. FACS results show mean fluorescence±S.D. for three experiments. Statistical significance of EGF compared with control of P

    Article Snippet: A BD Biosciences FACS III flow cytometer and software was used to analyse the FACS results.

    Techniques: Expressing, Microscopy, FACS, Fluorescence

    LPS and Con A-stimulated bovine PBMC. PBMC were separated from venous blood of one cow and immediately submitted to LPS and Con A stimulation assays, respectively. After three days in culture, non-adherent PBMC (lymphocytes) were fixed overnight at -20°C in 70% ethanol and stained with PI in the presence of RNAse for 30 min at room temperature. PBMC were analyzed in a Guava EasyCyte HT flow cytometer using software “Cell Cycle” (Merck Millipore). The percentages of S phase cells of control, LPS-stimulated and Con A-stimulated PBMC are shown as mean ± 1 standard deviation of three test replicates. The percentage of S phase cells was significantly higher after Con A stimulation (P

    Journal: PLoS ONE

    Article Title: Characterization of the blastogenic response to LPS of bovine peripheral blood mononuclear cells

    doi: 10.1371/journal.pone.0204827

    Figure Lengend Snippet: LPS and Con A-stimulated bovine PBMC. PBMC were separated from venous blood of one cow and immediately submitted to LPS and Con A stimulation assays, respectively. After three days in culture, non-adherent PBMC (lymphocytes) were fixed overnight at -20°C in 70% ethanol and stained with PI in the presence of RNAse for 30 min at room temperature. PBMC were analyzed in a Guava EasyCyte HT flow cytometer using software “Cell Cycle” (Merck Millipore). The percentages of S phase cells of control, LPS-stimulated and Con A-stimulated PBMC are shown as mean ± 1 standard deviation of three test replicates. The percentage of S phase cells was significantly higher after Con A stimulation (P

    Article Snippet: In this case, PBMC proliferation after 3 to 6 days was evaluated by CFSE staining [ ], or by staining nuclear antigen Ki-67 with fluorescein isothiocyanate (FITC)-Mouse anti-Ki-67 Set (BD Pharmingen, cat. 556026), according to the manufacturer’s directions, using a Guava EasyCyte HT flow cytometer with Incyte software.

    Techniques: Staining, Flow Cytometry, Cytometry, Software, Standard Deviation

    Cell growth assay. PBMC were separated from venous blood of two dairy cows, stained with CFSE and grown in medium supplemented with LPS (20 μg/mL final) or kept as unstimulated control in 25 cm 2 plastic bottles over 3 days at 39°C. After treatment with 0.5 mM EDTA in PBS, non-adherent PBMC (lymphocytes) were pelleted, resuspended in 0.2 mL of flow cytometry buffer and reacted with 10 μL of PI (50 micrograms/mL). After a 5-min incubation at 4°C, cells were analyzed in a Guava EasyCyte HT flow cytometry using the Cell Growth software, which discriminates CFSE and PI-stained cells. The gating dot plot of forward scatter versus green fluorescence allowed to select both resting and proliferating cells, and to discard debris from the analysis (left, panel A). Red-colored cells (panel A) were gated to the analysis dot plot (right). This included green (CFSE) versus red fluorescence (PI) with proper quadrant markers. Live proliferating cells (CFSE low, PI-negative) of a representative experiment are shown in the lower left quadrant. Similar results were obtained on lymphocytes of the other cow under study.

    Journal: PLoS ONE

    Article Title: Characterization of the blastogenic response to LPS of bovine peripheral blood mononuclear cells

    doi: 10.1371/journal.pone.0204827

    Figure Lengend Snippet: Cell growth assay. PBMC were separated from venous blood of two dairy cows, stained with CFSE and grown in medium supplemented with LPS (20 μg/mL final) or kept as unstimulated control in 25 cm 2 plastic bottles over 3 days at 39°C. After treatment with 0.5 mM EDTA in PBS, non-adherent PBMC (lymphocytes) were pelleted, resuspended in 0.2 mL of flow cytometry buffer and reacted with 10 μL of PI (50 micrograms/mL). After a 5-min incubation at 4°C, cells were analyzed in a Guava EasyCyte HT flow cytometry using the Cell Growth software, which discriminates CFSE and PI-stained cells. The gating dot plot of forward scatter versus green fluorescence allowed to select both resting and proliferating cells, and to discard debris from the analysis (left, panel A). Red-colored cells (panel A) were gated to the analysis dot plot (right). This included green (CFSE) versus red fluorescence (PI) with proper quadrant markers. Live proliferating cells (CFSE low, PI-negative) of a representative experiment are shown in the lower left quadrant. Similar results were obtained on lymphocytes of the other cow under study.

    Article Snippet: In this case, PBMC proliferation after 3 to 6 days was evaluated by CFSE staining [ ], or by staining nuclear antigen Ki-67 with fluorescein isothiocyanate (FITC)-Mouse anti-Ki-67 Set (BD Pharmingen, cat. 556026), according to the manufacturer’s directions, using a Guava EasyCyte HT flow cytometer with Incyte software.

    Techniques: Growth Assay, Staining, Flow Cytometry, Cytometry, Incubation, Software, Fluorescence

    Cell cycle assay. PBMC were separated from venous blood of two dairy cows and immediately submitted to a LPS stimulation assay in 25 cm 2 -flasks. After three days in culture, non-adherent PBMC (lymphocytes) were fixed overnight at -20°C in 70% ethanol and stained with PI in the presence of RNAse for 30 min at room temperature. PBMC were analyzed in a Guava EasyCyte HT flow cytometer using software “Cell Cycle” (Merck Millipore). Panel A: cells were analyzed by a combination of forward scatter (FSC) and PI incorporation. Red-colored cells were thus gated to the analysis histogram. Panel B: analysis histogram. Region 1, G1 phase cells; region 2, S phase cells; region 3, G2/M phase cells. The results obtained on one cow are shown, and these were similar to those of the other animal (see S1 Table ).

    Journal: PLoS ONE

    Article Title: Characterization of the blastogenic response to LPS of bovine peripheral blood mononuclear cells

    doi: 10.1371/journal.pone.0204827

    Figure Lengend Snippet: Cell cycle assay. PBMC were separated from venous blood of two dairy cows and immediately submitted to a LPS stimulation assay in 25 cm 2 -flasks. After three days in culture, non-adherent PBMC (lymphocytes) were fixed overnight at -20°C in 70% ethanol and stained with PI in the presence of RNAse for 30 min at room temperature. PBMC were analyzed in a Guava EasyCyte HT flow cytometer using software “Cell Cycle” (Merck Millipore). Panel A: cells were analyzed by a combination of forward scatter (FSC) and PI incorporation. Red-colored cells were thus gated to the analysis histogram. Panel B: analysis histogram. Region 1, G1 phase cells; region 2, S phase cells; region 3, G2/M phase cells. The results obtained on one cow are shown, and these were similar to those of the other animal (see S1 Table ).

    Article Snippet: In this case, PBMC proliferation after 3 to 6 days was evaluated by CFSE staining [ ], or by staining nuclear antigen Ki-67 with fluorescein isothiocyanate (FITC)-Mouse anti-Ki-67 Set (BD Pharmingen, cat. 556026), according to the manufacturer’s directions, using a Guava EasyCyte HT flow cytometer with Incyte software.

    Techniques: Cell Cycle Assay, Staining, Flow Cytometry, Cytometry, Software

    Magnetic separation with monoclonal antibodies. LPS-stimulated and BrDU-treated, as well as untreated PBMC of eight cows were frozen at -80°C. Next, they were thawed and separately reacted with one of the following monoclonal antibodies: IL-A30 (anti-bovine sIgM, B cell-specific), IL-A12 (anti-bovine CD4), IL-A51 (anti-bovine CD8), MM1A (anti-bovine CD3, pan-T). After 30 min at 4°C, cells were washed and 20 microliters of MACS anti-mouse IgG microbeads were added. After 15 minutes at 4°C, cells were reacted for a further 5 minutes with either PE or Alexa Fluor 488 anti-mouse IgG conjugates. Next, cells were submitted to magnetic separation on MACS MS columns according to the manufacturer’s directions. Cells were washed once, resuspended in 0.5 mL MACS separation buffer and analyzed in a Guava EasyCyte HT flow cytometer using Incyte software. Representative results of positive selections are shown. Unseparated cells stained with anti-IgG conjugates only served as negative control. On the whole, the assays were carried out on PBMC of eight cows and 16 samples, i.e. BrDU-treated and untreated PBMC of each cow.

    Journal: PLoS ONE

    Article Title: Characterization of the blastogenic response to LPS of bovine peripheral blood mononuclear cells

    doi: 10.1371/journal.pone.0204827

    Figure Lengend Snippet: Magnetic separation with monoclonal antibodies. LPS-stimulated and BrDU-treated, as well as untreated PBMC of eight cows were frozen at -80°C. Next, they were thawed and separately reacted with one of the following monoclonal antibodies: IL-A30 (anti-bovine sIgM, B cell-specific), IL-A12 (anti-bovine CD4), IL-A51 (anti-bovine CD8), MM1A (anti-bovine CD3, pan-T). After 30 min at 4°C, cells were washed and 20 microliters of MACS anti-mouse IgG microbeads were added. After 15 minutes at 4°C, cells were reacted for a further 5 minutes with either PE or Alexa Fluor 488 anti-mouse IgG conjugates. Next, cells were submitted to magnetic separation on MACS MS columns according to the manufacturer’s directions. Cells were washed once, resuspended in 0.5 mL MACS separation buffer and analyzed in a Guava EasyCyte HT flow cytometer using Incyte software. Representative results of positive selections are shown. Unseparated cells stained with anti-IgG conjugates only served as negative control. On the whole, the assays were carried out on PBMC of eight cows and 16 samples, i.e. BrDU-treated and untreated PBMC of each cow.

    Article Snippet: In this case, PBMC proliferation after 3 to 6 days was evaluated by CFSE staining [ ], or by staining nuclear antigen Ki-67 with fluorescein isothiocyanate (FITC)-Mouse anti-Ki-67 Set (BD Pharmingen, cat. 556026), according to the manufacturer’s directions, using a Guava EasyCyte HT flow cytometer with Incyte software.

    Techniques: Magnetic Cell Separation, Mass Spectrometry, Flow Cytometry, Cytometry, Software, Staining, Negative Control