Journal:

Article Title: CD47 is up-regulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis

doi: 10.1016/j.cell.2009.05.046

Figure Lengend Snippet: (A) GFP and human CD45 chimerism for mice transplanted with untransduced MOLM-13 cells (5×105 and either 5×105 Tet (n=6) or Tet-CD47 MOLM-13 (n=8) cells. (B) MOLM-13 chimerism in hematopoietic tissues was determined by human CD45 chimerism and measurement of tumour lesion size. (C) Hematoxylin and eosin sections of Tet-CD47 MOLM-13 transplanted liver (200×) (top panel). Periportal (arrow) and sinusoidal (arrowhead) tumor infilitration is evident. Examples of liver tumor formation and hepatomegaly in Tet-CD47 MOLM-13 transplanted mice versus control transplanted mice (middle panel). GFP fluorescence demonstrates tumor nodule formation as well diffuse infilitration (bottom panel). (D) 1×106 Tet (n=5) or Tet-CD47 MOLM-13 (n=4) cells were injected into the right femur of RAG2-/-, Gc-/- mice and the tissues were analyzed 50-75 days later for chimerism of MOLM-13 cells in BM. (E) Survival curve of mice transplanted intrafemorally with Tet or Tet-CD47 MOLM-13 cells. (F) Representative FACS plots from mice transplanted intrafemorally with Tet or Tet-CD47 MOLM-13 cells. R-right, L-left.

Article Snippet: Engraftment of MOLM-13 cells was assessed by using anti-human CD45 PE-Cy7 (Pharmingen), anti-mouse CD45.2 APC (clone AL1-4A2 produced in our lab), and anti-mouse CD47 Alexa-680 (mIAP301, produced in our lab).

Techniques: Fluorescence, Injection

Journal: International Journal of Molecular Sciences

Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

doi: 10.3390/ijms27114875

Figure Lengend Snippet: Positive and negative control cells for both marker panels. ( A ) HeLa (white arrows): in the first panel positive for cytokeratin (CK), Vimentin (Vim), VEGF and in the second panel positive for p16INK4A, negative for PD-L1 and CD45. ( B ) T98G (red arrows): positive for Vim. ( C ) CaSki (yellow arrows): positive for CK, Vim, VEGF, PD-L1 and p16INK4A. ( D ) MCF-7 (green arrows): positive for CK. All cell lines were negative for CD45. Only hematopoietic cells showed CD45 positive staining. Scale bar = 10 μm. Images were processed using Zeiss ZEN 3.7 software and the figure was assembled using Microsoft PowerPoint.

Article Snippet: In the second round of immunofluorescence staining, anti-human PD-L1 conjugated with AF 647 antibody (Cat. No. ab209960, abcam, Cambridge, UK), anti-human p16INK4a conjugated with AF488 antibody (Cat. No. ab199756, abcam, Cambridge, UK), and, for counterstaining, anti-human CD45 antibody conjugated with PE (Cat. No. 130-110-770, Miltenyi Biotech, Bergisch Gladbach, Germany) were applied, followed by image acquisition using the excitation filters mentioned above.

Techniques: Negative Control, Marker, Staining, Software

Journal: International Journal of Molecular Sciences

Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

doi: 10.3390/ijms27114875

Figure Lengend Snippet: Representative images of patient-derived DTCs with three different profiles. ( A ) DTC positive for Vim, VEGF and p16, negative for CK, PD-L1 and CD45. ( B ) CK and Vim positive while negative for VEGF, PD-L1, p16 and CD45. ( C ) DTC positive for Vim and PD-L1, negative for all other markers. Scale bar = 10 μm. Images were processed using Zeiss ZEN 3.7 software and figure was assembled using Microsoft PowerPoint.

Article Snippet: In the second round of immunofluorescence staining, anti-human PD-L1 conjugated with AF 647 antibody (Cat. No. ab209960, abcam, Cambridge, UK), anti-human p16INK4a conjugated with AF488 antibody (Cat. No. ab199756, abcam, Cambridge, UK), and, for counterstaining, anti-human CD45 antibody conjugated with PE (Cat. No. 130-110-770, Miltenyi Biotech, Bergisch Gladbach, Germany) were applied, followed by image acquisition using the excitation filters mentioned above.

Techniques: Derivative Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

doi: 10.3390/ijms27114875

Figure Lengend Snippet: Sequential multi-parameter immunofluorescent staining. After applying the first antibody panel (CK, Vim and VEGF), the slides were scanned and DTC profiles were assessed. Subsequently, releasable fluorochrome-conjugates were digested and the slides were subjected to the second antibody panel (PD-L1, p16, CD45) followed by scanning and DTC detection. Created with BioRender.com.

Article Snippet: In the second round of immunofluorescence staining, anti-human PD-L1 conjugated with AF 647 antibody (Cat. No. ab209960, abcam, Cambridge, UK), anti-human p16INK4a conjugated with AF488 antibody (Cat. No. ab199756, abcam, Cambridge, UK), and, for counterstaining, anti-human CD45 antibody conjugated with PE (Cat. No. 130-110-770, Miltenyi Biotech, Bergisch Gladbach, Germany) were applied, followed by image acquisition using the excitation filters mentioned above.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

doi: 10.3390/ijms27114875

Figure Lengend Snippet: CaSki cells before and after the release step. Representative immunofluorescence image of two CaSki cells spiked into bone marrow cells ( A ) before and ( B ) after the release step. The PE channel shows VEGF staining before staining with CD45, while nuclei were stained with DAPI (blue). Images were processed using Zeiss ZEN software and figure was assembled using Microsoft PowerPoint.

Article Snippet: In the second round of immunofluorescence staining, anti-human PD-L1 conjugated with AF 647 antibody (Cat. No. ab209960, abcam, Cambridge, UK), anti-human p16INK4a conjugated with AF488 antibody (Cat. No. ab199756, abcam, Cambridge, UK), and, for counterstaining, anti-human CD45 antibody conjugated with PE (Cat. No. 130-110-770, Miltenyi Biotech, Bergisch Gladbach, Germany) were applied, followed by image acquisition using the excitation filters mentioned above.

Techniques: Immunofluorescence, Staining, Software

Journal: Journal of Virology

Article Title: Control of Human T-Cell Leukemia Virus Type 1 (HTLV-1) Infection by Eliminating Envelope Protein-Positive Cells with Recombinant Vesicular Stomatitis Viruses Encoding HTLV-1 Primary Receptor

doi: 10.1128/JVI.01885-17

Figure Lengend Snippet: Blood cellularity changes in VSVΔG-NP-inoculated, HTLV-1-uninfected humanized mice. (A) A schematic drawing of the experimental schedule is shown. All NOJ mice were reconstituted with a human immune system by intrahepatic transplantation of human CD133 + hematopoietic stem cells into newborn mice. After humanization, mice were administered either medium (mock; n = 5) or G-complemented VSVΔG-NP (10 × 10 6 IU/body; n = 5) via a single i.p. injection. Arrowheads indicate the time points for blood collection. (B) Impact of rVSV inoculation on the blood cellularity of humanized mice. The absolute numbers of human lymphocytes are shown. Human CD45 + lymphocytes, CD19 + B cells, and CD4 or CD8 T cells were routinely analyzed by flow cytometry. The results are presented as the mean ± SD at each time point. Data collected over time from the same mock- or VSVΔG-NP-inoculated mice were compared at matched time points using a two-way ANOVA for repeated measures (NS, no statistical significance).

Article Snippet: For the staining of surface markers, anti-human CD45-phycoerythrin (PE)-Cy7, CD3-FITC, CD3-peridini chlorophyll protein (PerCP)-Cy5.5, CD4-PE, CD8-FITC, CD19-allophycocyanin (APC), and CD25-APC antibodies (Bay Bioscience Co., Ltd., Kobe, Japan) were used according to the manufacturer's protocol.

Techniques: Transplantation Assay, Injection, Flow Cytometry

Journal: Journal of Virology

Article Title: Control of Human T-Cell Leukemia Virus Type 1 (HTLV-1) Infection by Eliminating Envelope Protein-Positive Cells with Recombinant Vesicular Stomatitis Viruses Encoding HTLV-1 Primary Receptor

doi: 10.1128/JVI.01885-17

Figure Lengend Snippet: Antiviral effect of VSVΔG-NP on HTLV-1-infected humanized mice. (A) A schematic drawing of the experimental schedule is shown. After humanization, a total of 21 mice were inoculated intraperitoneally with HTLV-1-infected MT-2 cells lethally irradiated (at 0 dpi), and 15 of the mice were used for efficacy evaluation of VSVΔG-NP. HTLV-1-infected mice were administered either medium (mock; n = 5) or G-complemented rVSVs (10 × 10 6 IU/body), including VSVΔG ( n = 5) and VSVΔG-NP ( n = 5), by one-shot i.p. injection at 17 dpi. All coinfected mice were observed carefully during the experimental period and sacrificed at 56 dpi. One VSVΔG-inoculated mouse died at 41 dpi. Arrowheads indicate the time points for blood collection. (B and C) Therapeutic effects in the peripheral blood of HTLV-1-infected mice following rVSV inoculation. The frequencies of human lymphocytes are shown in panel B, and the absolute numbers of human lymphocytes are shown in panel C. Human CD45 + lymphocytes, total CD4 T cells, and CD25 + or CD25 − CD4 T cells were routinely analyzed by flow cytometry. CD3 + lymphocytes were gated to analyze the populations of CD4 T cells. The results are presented as the mean ± SD at each time point. (D) Quantification of HTLV-1 proviral copy numbers in the peripheral blood of infected mice. HTLV-1 PVL was determined by real-time PCR at 14, 28, 42, and 56 dpi. The HTLV-1 pX gene was determined as proviral DNA, and the human HBB gene was used as an internal reference gene. The absolute HTLV-1 proviral copy numbers per microliter of blood were calculated based on both the number of CD45 + cells and the PVL at each time point. One dot represents the result from an individual mouse, and the mean ± SD is shown. (E) Quantification of rVSV viral load in the plasma of coinfected mice. Total RNA was isolated from the plasma of each mouse at 14, 28, 42, and 56 dpi. Real-time RT-PCR was carried out to quantify the copy number of VSV-derived viral RNA targeting the L gene. The data are presented as log copies per milliliter of plasma. One dot represents the result from an individual mouse, and the mean ± SD is shown. Undetected samples are all indicated arbitrarily as 10 0 . (F) Kaplan-Meier curve of both mock- and VSVΔG-NP-inoculated mice. For survival analysis, a total of 6 mice were used and inoculated with medium or VSVΔG-NP as described above (mock, n = 3; VSVΔG-NP, n = 3). All coinfected mice were observed carefully until the experimental endpoint without blood collection. Statistical significance was determined by log rank test, and the resulting P value is shown. Asterisks in panels B to D represent significant differences between mock- versus VSVΔG-NP-inoculated mice (*, P < 0.05, **, P < 0.01, and NS, no statistical significance, by Mann-Whitney U test). Asterisks in panel E represent significant differences between VSVΔG-inoculated and VSVΔG-NP-inoculated mice (*, P < 0.05, and **, P < 0.01, by Mann-Whitney U test).

Article Snippet: For the staining of surface markers, anti-human CD45-phycoerythrin (PE)-Cy7, CD3-FITC, CD3-peridini chlorophyll protein (PerCP)-Cy5.5, CD4-PE, CD8-FITC, CD19-allophycocyanin (APC), and CD25-APC antibodies (Bay Bioscience Co., Ltd., Kobe, Japan) were used according to the manufacturer's protocol.

Techniques: Infection, Irradiation, Injection, Flow Cytometry, Real-time Polymerase Chain Reaction, Isolation, Quantitative RT-PCR, Derivative Assay, MANN-WHITNEY

Journal: Journal of Virology

Article Title: Control of Human T-Cell Leukemia Virus Type 1 (HTLV-1) Infection by Eliminating Envelope Protein-Positive Cells with Recombinant Vesicular Stomatitis Viruses Encoding HTLV-1 Primary Receptor

doi: 10.1128/JVI.01885-17

Figure Lengend Snippet: Elimination of HTLV-1 Env-expressing cells in the lymphoid tissues of VSVΔG-NP-inoculated mice. (A) Gene expression profiles of HTLV-1 Env in various organs of coinfected mice. Total RNA was isolated from the peripheral blood, bone marrow, spleen, mesenteric lymph nodes, lung, liver, and kidney of HTLV-1-infected mice following rVSV inoculation at 56 dpi (mock, n = 5, VSVΔG, n = 4, and VSVΔG-NP, n = 5, corresponding with to ). The expression levels of Env gene were determined by quantitative RT-PCR and were normalized to that of HPRT1. The mean value for the peripheral blood of mock controls is set to 1 as the reference for all specimens. Results are presented as the fold change compared with the reference (dotted line). (B) Quantification of rVSV viral load in various organs of coinfected mice. The total RNA specimens were described above. Real-time RT-PCR was performed to quantify the copy number of VSV-derived viral RNA targeting the L gene. Each dot represents the result from an individual mouse, and the mean ± SD is shown. (C) Cell surface expression of HTLV-1 gp46 was evaluated in the lymphoid tissues of coinfected mice. The bone marrow, spleen, mesenteric lymph nodes, and peripheral blood were harvested from the mice sacrificed at 56 dpi. The frequency of HTLV-1 gp46 + CD4 T cells among human CD45 + lymphocytes in the tissues was analyzed by flow cytometry. The percentages of HTLV-1 gp46 + CD4 T cells in mock-, VSVΔG-, or VSVΔG-NP-inoculated mice are shown. The results are presented as the mean ± SD. Asterisks in panels A and C represent significant differences between mock- versus VSVΔG-NP-inoculated mice (*, P < 0.05, **, P < 0.01, and NS, no statistical significance, by Mann-Whitney U test). Asterisks in panel B represent significant differences between VSVΔG-inoculated and VSVΔG-NP-inoculated mice (*, P < 0.05, and NS, no statistical significance, by Mann-Whitney U test).

Article Snippet: For the staining of surface markers, anti-human CD45-phycoerythrin (PE)-Cy7, CD3-FITC, CD3-peridini chlorophyll protein (PerCP)-Cy5.5, CD4-PE, CD8-FITC, CD19-allophycocyanin (APC), and CD25-APC antibodies (Bay Bioscience Co., Ltd., Kobe, Japan) were used according to the manufacturer's protocol.

Techniques: Expressing, Isolation, Infection, Quantitative RT-PCR, Derivative Assay, Flow Cytometry, MANN-WHITNEY

Journal: Journal of Cellular and Molecular Medicine

Article Title: Leucocyte and Platelet‐rich Fibrin: a carrier of autologous multipotent cells for regenerative medicine

doi: 10.1111/jcmm.13468

Figure Lengend Snippet: Antibodies used for flow cytometry analysis, Western blot and immunofluorescence

Article Snippet: PE mouse anti‐human CD45 , Santa Cruz Biotecnology, Inc.

Techniques: Flow Cytometry, Western Blot

Journal: bioRxiv

Article Title: Gravity-based microfiltration reveals unexpected prevalence of circulating tumor cell clusters in ovarian cancer

doi: 10.1101/773507

Figure Lengend Snippet: (A) Fluorescence images of OV-90 single cells and clusters captured using GµF and pump filtration using 8 µm filters. Cells were stained for cytokeratin (green) and CD45 (red). Nuclei were stained with DAPI (blue). Single cells (white arrows), small clusters and WBCs (red arrow) were captured using both configurations. Larger clusters, with > 5-6 cells were only captured with GµF. (B) Capture efficiency of clusters and single cells for GµF (green) and pump filtration (grey) at 0.1 and 0.5 mL min -1 . (C) Size distribution of the clusters before filtration and captured using GµF and pump filtration at ∼0.1 mL min -1 . For each replicate, a known number of OV-90 cells (∼120 clusters and ∼40 single cells) was spiked in diluted blood. Error bars correspond to the standard deviation of three replicated experiments. (D) Finite-element analysis comparing flow velocity in 8 µm pores for GµF and pump filtration at 0.1 mL min -1 , for 0% and 22% clogging. See also Tables S1 and S3.

Article Snippet: Anti-human CD45-PE (cluster of differentiation 45, Cat. #FAB1430P), anti-CK 18-AF 488 (cytokeratin 18, labeled with Alexa Fluor 488, Cat. #IC7619G) and anti-E-Cad (E-cadherin, Cat. #MAB18382, from mouse) were obtained from R&D systems.

Techniques: Fluorescence, Filtration, Staining, Standard Deviation

Journal: bioRxiv

Article Title: Gravity-based microfiltration reveals unexpected prevalence of circulating tumor cell clusters in ovarian cancer

doi: 10.1101/773507

Figure Lengend Snippet: (A) Fluorescence images of representative large and small clusters. Cells were stained for CK (green), CD45 (red) and with DAPI (blue). CTC-like cells (white arrow) are CK + /CD45 - /DAPI + and WBCs (red arrow) are CK - /CD45 + /DAPI + . (B) Capture efficiency depending on pore size, measured with OV-90 single cells (blue) and clusters (green) spiked in diluted blood. (C) Scatterplot and box plot of the size distribution of clusters captured by serial GµF through filters with deceasing pore size (28, 20, 15, 12, 10 and 8 µm) for three replicated experiments (n = 145, 300 and 550 spiked clusters). The boxes range from 25 th and 75 th percentiles, the whiskers correspond to 91 st and 9 th percentiles, and the horizontal lines represent the medians. (D) Cluster size distribution before filtration (dark grey, n = 80, 39 and 37) and after filtration at 0.1 ml min -1 and release by flowing PBS at either at 0.1 mL min -1 (light grey, n = 61, 30 and 35) or 1 mL min -1 (red, n = 37, 31 and 19). (E) Viablility of single cells (blue) and clusters (green) after filtration, rinsing, and release using complete OSE culture medium, PBS, or FBS at 4°C, at RT (22-23°C), and after 5 h incubation in low adhesion plates (no processsing) at 37°C. Error bars correspond to the standard deviation of three replicated experiments. p < 0.01: **; p < 0.05: *. See also Figure S3.

Article Snippet: Anti-human CD45-PE (cluster of differentiation 45, Cat. #FAB1430P), anti-CK 18-AF 488 (cytokeratin 18, labeled with Alexa Fluor 488, Cat. #IC7619G) and anti-E-Cad (E-cadherin, Cat. #MAB18382, from mouse) were obtained from R&D systems.

Techniques: Fluorescence, Staining, Pore Size, Filtration, Incubation, Standard Deviation