Journal: Annals of Gastroenterology : Quarterly Publication of the Hellenic Society of Gastroenterology

Article Title: Systemic levels of human β-defensin 1 are elevated in patients with cirrhosis

doi:

Figure Lengend Snippet: Elevated soluble CD14 (sCD14) concentration in the serum of cirrhotic patients. Concentration of sCD14 was measured in healthy controls (n=31), patients with chronic viral hepatitis (n=26) and patients with cirrhosis (n=50) as described in patients and methods. Data is presented graphically as Whisker box-plots

Article Snippet: For sCD14, human CD14 ELISA Development kit (R&D Systems, Abingdon, UK) was used following manufacturer’s instructions.

Techniques: Concentration Assay, Whisker Assay

Journal: Annals of Gastroenterology : Quarterly Publication of the Hellenic Society of Gastroenterology

Article Title: Systemic levels of human β-defensin 1 are elevated in patients with cirrhosis

doi:

Figure Lengend Snippet: High correlation between the levels of human beta defensin-1 (hBD-1) and soluble CD14 (sCD14) in the hepatic veins of cirrhotic patients . Concentrations of hBD-1 and sCD14 were measured as described in patients and methods. Each dot corresponds to individual patients with cirrhosis (n=45). Analysis was performed in samples collected from peripheral veins (n=25, ) and from hepatic veins (n=20, )

Article Snippet: For sCD14, human CD14 ELISA Development kit (R&D Systems, Abingdon, UK) was used following manufacturer’s instructions.

Techniques:

Journal: Annals of Gastroenterology : Quarterly Publication of the Hellenic Society of Gastroenterology

Article Title: Systemic levels of human β-defensin 1 are elevated in patients with cirrhosis

doi:

Figure Lengend Snippet: Soluble CD14 (sCD14) and lipopolysaccharide binding protein (LBP) strongly correlate in serum of patients with cirrhosis. Concentrations of sCD14 were measured as described in Patients and Methods. Concentrations of LBP were measured by a commercially available ELISA, according to manufacturer’s instructions. Each dot corresponds to individual patients with cirrhosis (n=36). Analysis was performed in samples collected from peripheral veins

Article Snippet: For sCD14, human CD14 ELISA Development kit (R&D Systems, Abingdon, UK) was used following manufacturer’s instructions.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: SARS-CoV-2 infection is associated with intestinal permeability, systemic inflammation, and microbial dysbiosis in hospitalized COVID-19 patients

doi: 10.1101/2023.12.07.570670

Figure Lengend Snippet: Plasma concentrations for I-FABP ( A ), Zonulin ( B ), LBP ( C ), and sCD14 ( D ) measured by ELISA and circulating fatty acids propionic acid ( E ), decanoic acid ( F ), butyric acid ( G ), nonanoic acid ( H ), and isovaleric acid ( I ) measured by LC-MS/MS. Comparisons between groups were performed by Kruskal-Wallis tests, followed by Dunn post-hoc tests if adjusted p values were below 0.05. Pairwise comparisons between each variable were corrected separately for false discovery rate by the Benjamini-Hochberg method and adjusted p values <0.05 were considered significant.

Article Snippet: Gut barrier damage biomarkers, including LBP (cell sciences CKH113), sCD14 (R&D Systems QK383), zonulin (MyBioSource MBS706368), and I-FABP (R&D Systems DFBP20) were measured using commercially available assays according to manufacturer’s guidelines.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Liquid Chromatography with Mass Spectroscopy

Journal: Frontiers in Immunology

Article Title: PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse

doi: 10.3389/fimmu.2022.1080501

Figure Lengend Snippet: Monocyte subpopulations` content in healthy individuals and CRC patients.

Article Snippet: For immunofluorescence (IF) staining, tumor FFPE clinical samples were treated with xylol solution and blocked with 3% BSA in PBS for 45 min, incubated with a combination of primary antibodies for 1,5 h; washed, and incubated with a combination of appropriate secondary antibodies for 45 min. Anti-PFKFB3 rabbit monоclonal antibody (1:50, #ab181861, Abcam, USA); anti-CD68 monoclonal mouse antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); anti-CD14 polyclonal sheep antibody (1:50, #BAF383, R&D Systems) were used.

Techniques:

Journal: Frontiers in Immunology

Article Title: PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse

doi: 10.3389/fimmu.2022.1080501

Figure Lengend Snippet: The distribution of CD163+ and CCR2+ peripheral blood monocytes in patients with colon and rectal cancers. Individual profiles of CCR2+ and CD163+ monocyte subsets for each patient with rectal and colon cancers. (A) , The distribution of monocytes of classical (CD14+CD16-), intermediate (CD14+CD16+) and non-classical (CD14-CD16+) populations expressing CCR2 (upper panel) and CD163 (lower panel) is demonstrated before and after NAC and after surgical resection in rectal cancer patients. (B) , The distribution of monocytes of classical (CD14+CD16-), intermediate (CD14+CD16+) and non-classical (CD14-CD16+) populations expressing CCR2 (upper panel) and CD163 (lower panel) is demonstrated before and after surgical resection in colon cancer patients. (C) , Associations of CCR2-expressing monocyte subsets with hematogenous and lymphatic metastasis in rectal cancer patients. (D) , Associations of CD163-expressing monocyte subsets with hematogenous and lymphatic metastasis in colon cancer patients. M 0 , metastasis-negative status, M 1 , metastasis-positive status. N 0 , lymph node-negative status, N 1-3 , lymph node-positive status.

Article Snippet: For immunofluorescence (IF) staining, tumor FFPE clinical samples were treated with xylol solution and blocked with 3% BSA in PBS for 45 min, incubated with a combination of primary antibodies for 1,5 h; washed, and incubated with a combination of appropriate secondary antibodies for 45 min. Anti-PFKFB3 rabbit monоclonal antibody (1:50, #ab181861, Abcam, USA); anti-CD68 monoclonal mouse antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); anti-CD14 polyclonal sheep antibody (1:50, #BAF383, R&D Systems) were used.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse

doi: 10.3389/fimmu.2022.1080501

Figure Lengend Snippet: An activator of glycolysis PFKFB3 is overexpressed in colon cancer and is indicative for higher risk of tumor relapse in colon cancer but not rectal cancer. (A) , Сolon cancer tissue is massively infiltrated by PFKFB3-positive monocytes. IF/confocal microscopy analysis was performed for 10 colon tumor tissues. The infiltration of CD14+CD68+PFKFB3+ cells was found in all samples. Representative image is given from one patient. Scale bar corresponds to 50 µm in main image and 20 µm in zoom image. (B) , Spearman correlation coefficients between PFKFB3 expression, M2 macrophage gene expressions and predicted cell abundance scores, FDR<0.05. (C) , Predicted cell composition of CD45+ AOIs and hierarchical clustering of AOIs. (D) , Difference in monocyte and macrophage cell abundance scores between the CD45+ AOIs in colon and rectal cancers (the Mann-Whitney U test was applied). (E) , PFKFB3 gene expression is elevated in patients with recurrence and larger tumor size in colon cancer. Variance in PFKFB3 expression was stabilized via the variance stabilizing transformation (VST). (F) , PFKFB3 had prognostic significance for the DFS and OS. High-risk group had worse survival rates compared to low-risk group. ROC analysis and Kaplan–Meier method were applied.

Article Snippet: For immunofluorescence (IF) staining, tumor FFPE clinical samples were treated with xylol solution and blocked with 3% BSA in PBS for 45 min, incubated with a combination of primary antibodies for 1,5 h; washed, and incubated with a combination of appropriate secondary antibodies for 45 min. Anti-PFKFB3 rabbit monоclonal antibody (1:50, #ab181861, Abcam, USA); anti-CD68 monoclonal mouse antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); anti-CD14 polyclonal sheep antibody (1:50, #BAF383, R&D Systems) were used.

Techniques: Confocal Microscopy, Expressing, MANN-WHITNEY, Gene Expression, Transformation Assay

Journal: Frontiers in immunology

Article Title: The significance of CD16+ monocytes in the occurrence and development of chronic thromboembolic pulmonary hypertension: insights from single-cell RNA sequencing.

doi: 10.3389/fimmu.2024.1446710

Figure Lengend Snippet: FIGURE 2 Changes in peripheral blood monocyte subsets in CTEPH patients. (A) UMAP plot of Mononuclear Phagocyte System (MPS) was divided into three cell types: monocytes, macrophages, and conventional dendritic cells (cDCs). Subsequently, monocytes were further divided into CD14+ monocytes and CD16+ monocytes. (B) A dot plot was used to annotate marker genes for CD14+ monocytes and CD16+ monocytes, along with a heatmap displaying the top 10 differentially expressed genes between the two cell types. (C) Ro/e (ratio of observed cell number to expected cell number) revealed the proportion of CD16+ monocytes in the CTEPH-N, CTEPH-I and HC groups. (D) The heat map showed the degree of correlation, and the numbers in the graph represented the correlation coefficient. (6MWD: 6-minute walk distance; PVR: pulmonary vascular resistance) (E) Schematic diagram of the gating strategy for distinguishing CD14+ monocytes from CD16+ monocytes by flow cytometry. (F) Differences in the proportion of CD16+ monocyte subsets in total monocytes between CTEPD patients (n = 15) and matched healthy controls (n = 15) were assessed by flow cytometry. All data were presented as means ± SEM, ***P < 0.001.

Article Snippet: Then the cells were resuspend in the flow cytometer wash buffer (2% FBS in PBS) and stained with the following antibodies according to the standard protocol: CD14-PE (Elabscience, E-ABF1209D) and CD16-FITC (Elabscience, E-AB-F1236C) for monocyte labeling.

Techniques: Marker, Cytometry

Journal: Frontiers in immunology

Article Title: The significance of CD16+ monocytes in the occurrence and development of chronic thromboembolic pulmonary hypertension: insights from single-cell RNA sequencing.

doi: 10.3389/fimmu.2024.1446710

Figure Lengend Snippet: FIGURE 3 The functional characteristics of peripheral blood CD16+ monocytes in CTEPH patients. (A) GO analysis (biological process) of upregulated genes in CD16+ monocytes compared with CD14+ monocytes in CTEPH patients. (B) KEGG analysis of upregulated genes in CD16+ monocytes compared with CD14+ monocytes in CTEPH patients. (C) GSEA bar plot of CD16+ monocytes versus CD14+ monocytes in patients with CTEPH. (D) GO analysis (biological process) of upregulated genes in CD16+ monocytes between the CTEPH and healthy control samples. (E) KEGG analysis of upregulated genes in CD16+ monocytes between CTEPH patients and healthy controls. (F) GSVA heatmap of CD16+ monocytes between CTEPH patients and healthy controls. (G) Heat map of transcription factors upregulated in CD16+ monocytes between CTEPH patients and healthy controls.

Article Snippet: Then the cells were resuspend in the flow cytometer wash buffer (2% FBS in PBS) and stained with the following antibodies according to the standard protocol: CD14-PE (Elabscience, E-ABF1209D) and CD16-FITC (Elabscience, E-AB-F1236C) for monocyte labeling.

Techniques: Functional Assay, Control

Journal: Scientific reports

Article Title: Blood-brain barrier and gut barrier dysfunction in chronic kidney disease with a focus on circulating biomarkers and tight junction proteins.

doi: 10.1038/s41598-022-08387-7

Figure Lengend Snippet: Figure 2. Analysis of soluble CD14 (sCD14) levels in haemodialysis (HD) and non-CKD controls (control). (A) Soluble CD14 levels. The number of samples (n) that each analyte was detected in is noted for each group. Data presented at median and interquartile range. Statistical significance, ***p < 0.001, ****p < 0.0001. (B) Spearman’s correlation between sCD14 and trimethylamine N-oxide (TMAO) levels.

Article Snippet: The S100B ELISA kit was performed in accordance with manufacturer guidelines, with the alteration that samples were undiluted. sCD14 levels in HD patients was previously recorded and reported87, for measurements in non-CKD controls the same CD14 ELISA kit (DC140, R&D Systems, UK) was used, and run in accordance with manufacturer guidelines.

Techniques: Control

Journal: Scientific reports

Article Title: Blood-brain barrier and gut barrier dysfunction in chronic kidney disease with a focus on circulating biomarkers and tight junction proteins.

doi: 10.1038/s41598-022-08387-7

Figure Lengend Snippet: Figure 1. Analysis of serum biomarkers in haemodialysis (HD) and non-CKD controls (control). (A) Brain-derived neurotrophic factor (BDNF), (B) Neuron-specific enolase (NSE) were measured by enzyme- linked immunosorbent assay (ELISA). (C) Trimethylamine N-oxide (TMAO) was measured by liquid chromatography-mass spectrometry (LC–MS). The number of samples (n) that each analyte was detected in is noted for each group. Data presented at median and interquartile range. Statistical significance, ***p < 0.001, ****p < 0.0001. (D) Spearman’s correlation between BDNF and TMAO levels. (E) Spearman’s correlation between NSE and TMAO levels.

Article Snippet: The S100B ELISA kit was performed in accordance with manufacturer guidelines, with the alteration that samples were undiluted. sCD14 levels in HD patients was previously recorded and reported87, for measurements in non-CKD controls the same CD14 ELISA kit (DC140, R&D Systems, UK) was used, and run in accordance with manufacturer guidelines.

Techniques: Control, Derivative Assay, Enzyme-linked Immunosorbent Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal:

Article Title: LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 Expression in Human Lymphatic Endothelium

doi: 10.1369/jhc.7A7299.2007

Figure Lengend Snippet: Expression of toll-like receptor 4 (TLR4), MD-2, and CD14. (A) Immunohistochemical analysis. The cell membrane was visualized with rhodamine-conjugated Con A (Con A, in red), and the reaction products to antibodies for TLR4, MD-2, and CD14 were visualized with an AF488-conjugated second antibody (AF488, in green). Merged images indicate that the expression of TLR4, MD-2, and CD14 was immunohistochemically observed in the cytoplasm and on the cell membrane of LEC, and that the three were detected on the membrane of HEK-293 cells stably transfected with the human TLR4, MD-2, and CD14 genes (tHEK), and not in the negative control HEK-293 cells (HEK). Bar = 100 μm. (B) RT-PCR analysis. RT-PCR products for TLR4, MD-2, and CD14 mRNAs were not detected in HEK-293 but were detected in LEC and in tHEK. MW, molecular weight marker.

Article Snippet: The cells were immunostained with 1 μg/ml of rabbit antiserum to human Prox1 (AngioBio Co.; Del Mar, CA) and of monoclonal antibodies to human podoplanin (AngioBio), VCAM-1, and ICAM-1 (R and D Systems, Inc.; Minneapolis, MN), and also immunostained with 5 μg/ml of monoclonal antibodies to TLR4, MD-2, and CD14 (R and D Systems).

Techniques: Expressing, Immunohistochemical staining, Membrane, Stable Transfection, Transfection, Negative Control, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Marker

Journal:

Article Title: LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 Expression in Human Lymphatic Endothelium

doi: 10.1369/jhc.7A7299.2007

Figure Lengend Snippet: Expression of genes for lymphatic endothelial markers, and TLR-associated and leukocyte adhesion molecules in human neonatal dermal lymphatic microvascular endothelial cells with LPS treatments

Article Snippet: The cells were immunostained with 1 μg/ml of rabbit antiserum to human Prox1 (AngioBio Co.; Del Mar, CA) and of monoclonal antibodies to human podoplanin (AngioBio), VCAM-1, and ICAM-1 (R and D Systems, Inc.; Minneapolis, MN), and also immunostained with 5 μg/ml of monoclonal antibodies to TLR4, MD-2, and CD14 (R and D Systems).

Techniques: Expressing, Control, Marker, Activation Assay

Journal: Cancer Reports

Article Title: The effects of CD148 Q276P / R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: Generation of A431D cells that stably express CD148 forms (WT, Q276P/R326Q). (A) DNA sequence of mutated CD148 cDNA, confirming Q276P and R326Q mutations. (B) CD148 surface expression were examined in A431D‐CD148 (WT, Q276P/R326Q) cells and the control A431D cells that was generated by the infection of empty retrovirus (Mock) by flow cytometry using a PE‐conjugated anti‐CD148 antibody. Representative results of four independent experiments are shown. Mean fluorescence intensity is also shown. The fluorescence intensity of CD148 WT cells is expressed as 1.0. A431D‐CD148 WT and Q276P/R326Q cells express comparable levels of CD148. A431D‐Mock cells show no CD148 expression. (C) Expression of CD148 in A431D‐CD148 (WT, Q276P/R326Q) cells was also examined by Western blotting using anti‐CD148 or anti‐HA antibodies. Fifty micrograms of protein cell lysates were loaded into each well. The loading of proteins was assessed by anti‐β actin Western blot. The ratios of CD148 or HA to β actin are also shown. The ratios in A431D‐CD148 WT cells are expressed as 1.0. Representative results of four independent experiments are shown. (D) Immunolocalization of CD148 or HA epitope (both red) were examined in A431D‐Mock and A431D‐CD148 (WT, Q276P/R326Q) cells. The antibody against CD148 extracellular domain was used. Representative results of four independent experiments are shown. Scale bar = 25 μm. CD148 WT and Q276P/R326Q are similarly localized to the cell membrane (arrowheads). In anti‐HA immunostaining, there is perinuclear CD148 presence that is generally observed in stable cells

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSA‐PBS, and 2 × 10 5 cells were incubated with 5 μl of PE‐conjugated anti‐human CD148 mouse monoclonal (clone 143‐41, R&D Systems) or 10 μl of PE‐conjugated anti‐human EGFR (BD Biosciences) for 45 min at 4°C.

Techniques: Stable Transfection, Sequencing, Expressing, Control, Generated, Infection, Flow Cytometry, Fluorescence, Western Blot, Membrane, Immunostaining

Journal: Cancer Reports

Article Title: The effects of CD148 Q276P / R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: Cell proliferation rate in A431D‐CD148 (WT, Q276P/R326Q) cells. Cells (1.0 × 10 3 ) were plated in 96‐well plates and serum starved (0.1% FBS) for overnight (day 1). Cells were then cultured in growth medium supplemented with 2.5% FBS. Cell number was assessed at day 1, 2, 3 and 4. Data are means ± SEM of quadruplicate determinations. Representative data of four independent experiments is shown. ** p < .01. A431D‐CD148 (WT, Q276P/R326Q) cells showed lower cell proliferation rates than A431D‐Mock cells. No significant difference was observed between A431D‐CD148 WT and Q276P/R326Q cells

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSA‐PBS, and 2 × 10 5 cells were incubated with 5 μl of PE‐conjugated anti‐human CD148 mouse monoclonal (clone 143‐41, R&D Systems) or 10 μl of PE‐conjugated anti‐human EGFR (BD Biosciences) for 45 min at 4°C.

Techniques: Cell Culture

Journal: Cancer Reports

Article Title: The effects of CD148 Q276P / R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: Expression of epidermal growth factor receptor (EGFR) and EGF‐induced ERK phosphorylation in A431D‐CD148 (WT, Q276P/R326Q) cells. (A) Cell surface expression of EGFR in A431D‐CD148 (WT and Q276P/R326Q) and A431D‐Mock (control) cells were examined by flow cytometry using a PE‐conjugated anti‐human EGFR antibody. HEK293 cells that do not express EGFR were used as a negative control. Mean fluorescence intensity is also shown (bottom right). The fluorescence intensity of A431D‐Mock cells is expressed as 1.0. Representative results of three independent experiments are shown. All three A431D cells showed comparable level of EGFR expression. (B) EGF‐induced ERK1/2 phosphorylation was examined in A431D‐CD148 (WT and Q276P/R326Q) and A431D‐Mock cells by ELISA as described in Section . Cells were treated with 10 ng/ml EGF for 5, 10, 15, and 30 min. The cells cultured in serum starved (SS) condition (0.1% FBS medium) for 30 min were used as a control. The p‐ERK/ERK ratios were normalized to serum‐starved (30 min) A431D‐Mock cells. Representative results of five independent experiments are shown. Data are means ± SEM of quadruplicate measurements. *** p < .001

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSA‐PBS, and 2 × 10 5 cells were incubated with 5 μl of PE‐conjugated anti‐human CD148 mouse monoclonal (clone 143‐41, R&D Systems) or 10 μl of PE‐conjugated anti‐human EGFR (BD Biosciences) for 45 min at 4°C.

Techniques: Expressing, Phospho-proteomics, Control, Flow Cytometry, Negative Control, Fluorescence, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cancer Reports

Article Title: The effects of CD148 Q276P / R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: Immunoblotting to assess EGF‐induced epidermal growth factor receptor (EGFR) and ERK1/2 phosphorylation in A431D‐CD148 (WT, Q276P/R326Q) cells. (A) and (B) EGF‐induced EGFR and ERK1/2 phosphorylation was examined in A431D‐CD148 (WT, Q276P/R326Q) and A431D‐Mock cells by Western blotting as described in Section . Cells were plated in 100 mm dishes, serum was reduced (0.5% FBS), then cells were treated with 10 ng/ml EGF for 5, 10, 15, 30 min. The cells cultured in serum starved (SS) condition (0.1% FBS medium) for 30 min were used as a negative control. The p‐EGFR/EGFR ratios were normalized to A431D‐Mock cells treated with EGF for 5 min as EGFR phosphorylation was undetectable in serum starved (0.1% FBS) cells. The p‐ERK/ERK ratios were normalized to serum‐starved A431D‐Mock cells. Representative results of five independent experiments are shown

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSA‐PBS, and 2 × 10 5 cells were incubated with 5 μl of PE‐conjugated anti‐human CD148 mouse monoclonal (clone 143‐41, R&D Systems) or 10 μl of PE‐conjugated anti‐human EGFR (BD Biosciences) for 45 min at 4°C.

Techniques: Western Blot, Phospho-proteomics, Cell Culture, Negative Control

Journal: Cancer Reports

Article Title: The effects of CD148 Q276P / R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: EGF‐induced cell proliferation in A431D‐CD148 (WT, Q276P/R326Q) cells. Cells (1.2 × 10 3 ) were plated in 96‐well plates (day 0), then serum starved (0.1% FBS) for overnight (day 1). Cells were then treated with 60 ng/ml, 120 ng/ml, and 240 ng/ml EGF in growth medium supplemented with 0.3% FBS. Cell number was assessed at day 1 (before EGF stimulation) and day 3. Representative results of four independent experiments are shown. Data are means ± SEM of quadruplicate determinations. ** p < .01, * p < .05

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSA‐PBS, and 2 × 10 5 cells were incubated with 5 μl of PE‐conjugated anti‐human CD148 mouse monoclonal (clone 143‐41, R&D Systems) or 10 μl of PE‐conjugated anti‐human EGFR (BD Biosciences) for 45 min at 4°C.

Techniques:

Journal: Cancer Reports

Article Title: The effects of CD148 Q276P / R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: Effects of TSP1 on cell proliferation in A431D‐CD148 (WT, Q276P/R326Q) cells. Cells (1.0 × 10 3 ) were plated in 96‐well plates (day 0), then serum starved (0.1% FBS) for overnight (day 1). Cells were then treated with 1, 10, 20 nM of TSP1 in growth medium supplemented with 2.5% FBS. Cell number was assessed at day 1 (before TSP1 stimulation) and day 4. Representative results of four independent experiments are shown. Data are means ± SEM of quadruplicate determinations. ** p < .01

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSA‐PBS, and 2 × 10 5 cells were incubated with 5 μl of PE‐conjugated anti‐human CD148 mouse monoclonal (clone 143‐41, R&D Systems) or 10 μl of PE‐conjugated anti‐human EGFR (BD Biosciences) for 45 min at 4°C.

Techniques:

Journal: Pediatric Rheumatology Online Journal

Article Title: Surface expression and genotypes of Toll- like receptors 2 and 4 in patients with juvenile idiopathic arthritis and systemic lupus erythematosus

doi: 10.1186/1546-0096-11-9

Figure Lengend Snippet: Representative flow cytometry histograms showing (a) TLR2 and (b) TLR4 expression on CD14 + monocytes. Blue filled histograms: isotype controls and green or red filled histogram TLR expression. Mean channel fluorescence intensity (MFI) derived from fluorescence histogram was used to study the level of cell surface TLR expression. Delta MFI (dMFI) was calculated as a subtraction and recorded as the MFI of the TLR2 or TLR4 antibody minus the MFI of the isotype-matched control antibody.

Article Snippet: For surface staining 100 μl of whole blood samples were incubated with the following anti-human primary antibodies for 15 minutes in the dark at 4°C: anti-CD14 monoclonal antibody (mAb; 10 μl per 100 μl blood, FITC or PE labelled, clone 8 G3; Diaclone, Besançon, France), anti-TLR2 mAb (10 μl, FITC labelled, clone HTA 125; eBioscience, San Diego, CA) or TLR4 mAb (10 μl, PE labelled, clone HTA 125; eBioscience, San Diego, CA).

Techniques: Flow Cytometry, Expressing, Fluorescence, Derivative Assay, Control

Journal: Pediatric Rheumatology Online Journal

Article Title: Surface expression and genotypes of Toll- like receptors 2 and 4 in patients with juvenile idiopathic arthritis and systemic lupus erythematosus

doi: 10.1186/1546-0096-11-9

Figure Lengend Snippet: (a) Mean fluorescence intensity (dMFI) of TLR2-expression on CD14 + monocytes of patients with JIA, SLE and healthy controls. (b) Mean fluorescence intensity (dMFI) of TLR4-expression on CD14 + monocytes of patients with JIA, SLE and healthy controls.

Article Snippet: For surface staining 100 μl of whole blood samples were incubated with the following anti-human primary antibodies for 15 minutes in the dark at 4°C: anti-CD14 monoclonal antibody (mAb; 10 μl per 100 μl blood, FITC or PE labelled, clone 8 G3; Diaclone, Besançon, France), anti-TLR2 mAb (10 μl, FITC labelled, clone HTA 125; eBioscience, San Diego, CA) or TLR4 mAb (10 μl, PE labelled, clone HTA 125; eBioscience, San Diego, CA).

Techniques: Fluorescence, Expressing

Journal: Pediatric Rheumatology Online Journal

Article Title: Surface expression and genotypes of Toll- like receptors 2 and 4 in patients with juvenile idiopathic arthritis and systemic lupus erythematosus

doi: 10.1186/1546-0096-11-9

Figure Lengend Snippet: (a) Mean fluorescence intensity (dMFI) of TLR2-expression on CD14 + monocytes of patients in various disease phases compared to TLR2-expression on monocytes of healthy controls. (b) Mean fluorescence intensity (dMFI) of TLR4-expression on CD14 + monocytes of patients in various disease phases compared to TLR4-expression on monocytes of healthy controls.

Article Snippet: For surface staining 100 μl of whole blood samples were incubated with the following anti-human primary antibodies for 15 minutes in the dark at 4°C: anti-CD14 monoclonal antibody (mAb; 10 μl per 100 μl blood, FITC or PE labelled, clone 8 G3; Diaclone, Besançon, France), anti-TLR2 mAb (10 μl, FITC labelled, clone HTA 125; eBioscience, San Diego, CA) or TLR4 mAb (10 μl, PE labelled, clone HTA 125; eBioscience, San Diego, CA).

Techniques: Fluorescence, Expressing