anti-human garp antibody  (R&D Systems)

Journal: Cancers

Article Title: Spatial Distribution of Non-Immune Cells Expressing Glycoprotein A Repetitions Predominant in Human and Murine Metastatic Lymph Nodes

doi: 10.3390/cancers15235621

Figure Lengend Snippet: Evaluation of GARP in HUVEC, LEC, and HLF cells cultured under basal conditions. ( a ) Western blot analysis of GARP expression. The blot is a representative blot out of 4 independent experiments. The bar graph shows the quantification of GARP protein levels relative to the GAPDH protein signal (GARP/GAPDH signals) ( n = 4, means ± SD, n.s. determined by one-way ANOVA). ( b ) Flow cytometry analysis of GARP at the surface of primary cells. Jurkat cells overexpressing GARP (Jurkat−hGARP) were used as a positive control. The isotype control is represented in grey, and the positive signal is depicted in red as a percentage of the maximum. The relative MFI of GARP in flow cytometry is represented with a bar graph ( n ≥ 3, means ± SD, n.s., no significance, determined by one-way ANOVA).

Article Snippet: Sections were blocked with animal-free blocking solution (15019 L, Cell Signaling), followed by an incubation with either 5 µg/mL of mouse monoclonal anti-human GARP antibody (clone MHG-6 [ ]) in Dako antibody diluent or no primary antibody (negative control) for 90 min. After two washes with PBS supplemented with Tween20 (PBS-T), the EnVision-HRP secondary antibody (K4001, Dako Agilent, Diegem, Belgium) was incubated at room temperature for 30 min. Staining was amplified using tyramide signal amplification working solution (TSA, NEL741001KT, PerkinElmer) containing fluorescein isothiocyanate dye (1:500, FITC) for 10 min, washed thrice with PBS-T.

Techniques: Cell Culture, Western Blot, Expressing, Flow Cytometry, Positive Control

Journal: Cancers

Article Title: Spatial Distribution of Non-Immune Cells Expressing Glycoprotein A Repetitions Predominant in Human and Murine Metastatic Lymph Nodes

doi: 10.3390/cancers15235621

Figure Lengend Snippet: Full scans of Western blot images shown in a. ( a ) Western blot membrane of GARP in Jurkat hGARP used as a positive control. The red rectangle corresponds to the first lane in a. ( b ) Western blot membrane of GARP on HUVECs, LECs, and HLFs in which the red rectangle corresponds to the right part of Western blot shown in a.

Article Snippet: Sections were blocked with animal-free blocking solution (15019 L, Cell Signaling), followed by an incubation with either 5 µg/mL of mouse monoclonal anti-human GARP antibody (clone MHG-6 [ ]) in Dako antibody diluent or no primary antibody (negative control) for 90 min. After two washes with PBS supplemented with Tween20 (PBS-T), the EnVision-HRP secondary antibody (K4001, Dako Agilent, Diegem, Belgium) was incubated at room temperature for 30 min. Staining was amplified using tyramide signal amplification working solution (TSA, NEL741001KT, PerkinElmer) containing fluorescein isothiocyanate dye (1:500, FITC) for 10 min, washed thrice with PBS-T.

Techniques: Western Blot, Membrane, Positive Control

Journal: PLoS ONE

Article Title: Impaired Circulating CD4 + LAP + Regulatory T Cells in Patients with Acute Coronary Syndrome and Its Mechanistic Study

doi: 10.1371/journal.pone.0088775

Figure Lengend Snippet: Bood samples were collected from patients with CPS (n = 20), CSA (n = 17) and ACS (n = 37, AMI (n = 18), and UA (n = 19)). PBMCs were freshly isolated or stimulated with CD3/CD28 for 24 h. Then the cells were stained with anti-human CD4-FITC, anti-human GARP-PE and analyzed by flow cytometry using FACS Calibur (BD). (A) Representative dot plot shows the gated CD4 + T cells on the FSC/SSC. (B) Representative FACS images show GARP expression on CD4 + T cells in unstimulated PMBCs (upper panel) and stimulated PBMCs (lower panel) from one patient in each group. Comparison of the CD4 + GARP + T cells frequencies in unstimulated PBMCs (C) and stimulated PBMCs (D) among four groups. * p <0.01 vs. CSA or CPS; # p >0.05 vs. CPS.

Article Snippet: After 24 hours, the cells were harvested and stained with anti-human LAP-APC (clone 27232, R&D Systems), anti-human CD4-FITC (R&D Systems), and anti-human GARP-PE (G14D9, eBioscience) for 30 min at 4°C.

Techniques: Isolation, Staining, Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: Impaired Circulating CD4 + LAP + Regulatory T Cells in Patients with Acute Coronary Syndrome and Its Mechanistic Study

doi: 10.1371/journal.pone.0088775

Figure Lengend Snippet: Bood samples were collected from patients with ACS (AMI (n = 10) and UA (n = 10)) and controls (CSA (n = 17), CPS (n = 20), and PBMCs were freshly isolated or stimulted with CD3/CD28 for 24 h, then the cells were stained with anti-human CD4-FITC,anti-human LAP-APC, anti-human GARP-PE and analyzed the data by flow cytometry using FACS Calibur (BD).(A) Representative dot plot shows the gated CD4 T cells on the FSC/SSC. (B) Representative FACS pictures show the GARP and LAP expreesion on CD4 + T cells in unstimulated PMBCs (upper panel) and stimulated PBMCs (lower panel) from one patient in each group. (C) Statistical analysis of the percentage CD4 + LAP + GARP + T cells. Dark bars indicate data from unstimulated samples. Hatched bars indicate data from stimulated samples. * p <0.05 vs. CSA or CPS; # p >0.05 vs.CPS.

Article Snippet: After 24 hours, the cells were harvested and stained with anti-human LAP-APC (clone 27232, R&D Systems), anti-human CD4-FITC (R&D Systems), and anti-human GARP-PE (G14D9, eBioscience) for 30 min at 4°C.

Techniques: Isolation, Staining, Flow Cytometry

Journal: Biomedicines

Article Title: GARP Regulates the Immune Capacity of a Human Autologous Platelet Concentrate.

doi: 10.3390/biomedicines10123136

Figure Lengend Snippet: Figure 1. Glycoprotein A repetitions predominant (GARP) is expressed on the surface of platelets and exists as a soluble factor found in liquid platelet rich fibrin (iPRF). (A,B) iPRF was stained directly for the platelet marker, CD41a, and GARP and underwent flow cytometric analysis. Isotype antibodies were used as a control. (A) Dot blots show one representative result. (B) Bar diagrams of GARP expression show the pooled data of percentages (%) of GARP positive platelets in iPRF and the mean fluorescence intensity (MFI) of GARP (n = 3 ± SD, analyzed by paired Students T-tests). (C) Expression of GARP in iPRF was analyzed via Western blot. iPRF lysate was loaded in increasing amounts. Recombinant soluble GARP (sGARP) was loaded at 1 ng/lane and served as a positive control, whereas β-actin acted as the loading control. (D) Bar graph shows the soluble GARP (sGARP) levels in peripheral blood and iPRF analyzed via ELISA (n = 3 ± SD, ns determined by paired Students T-tests).

Article Snippet: Labeled CD4+ T cells were plated in 48 well plates at 106 cells/mL in X-Vivo 15 (Lonza, #BE02-060F, Basel, Switzerland) and incubated for 3 days in the presence or absence of platelets derived from iPRF at a ratio of 1:50 as well as 10 μg/mL anti-GARP Ab (Origene) before undergoing flow cytometric analysis.

Techniques: Staining, Marker, Control, Expressing, Western Blot, Recombinant, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: GARP Regulates the Immune Capacity of a Human Autologous Platelet Concentrate.

doi: 10.3390/biomedicines10123136

Figure Lengend Snippet: Figure 2. Isolated platelets from liquid platelet rich fibrin (iPRF) induce a regulatory phenotype in CD4+ T cells. CD4+ T cells and platelets (Plt.) were cocultured at a ratio of 1 to 50 and were treated with 10 µg/mL anti-GARP antibody (Ab). CD4+ T cells were prelabled with carboxyfluorescein succinimidyl ester (CFSE) to track their proliferation and stimulated with 0.5 µg/mL anti-CD3 and 1.0 µg/mL anti-CD28 Abs in the presence or absence of platelets. (A–C) The expression of

Article Snippet: Labeled CD4+ T cells were plated in 48 well plates at 106 cells/mL in X-Vivo 15 (Lonza, #BE02-060F, Basel, Switzerland) and incubated for 3 days in the presence or absence of platelets derived from iPRF at a ratio of 1:50 as well as 10 μg/mL anti-GARP Ab (Origene) before undergoing flow cytometric analysis.

Techniques: Isolation, Expressing

Journal: Biomedicines

Article Title: GARP Regulates the Immune Capacity of a Human Autologous Platelet Concentrate.

doi: 10.3390/biomedicines10123136

Figure Lengend Snippet: Figure 4. Liquid platelet rich fibrin (iPRF) polarizes M0-macrophages in a more “M0/M2-like” phenotype through a GARP independent mechanism. Human monocyte derived macrophages were polarized, treated with 400 µL iPRF/well, 10 µg/mL anti-GARP antibody (Ab) or were left untreated for 2 days as indicated. Surface marker expression was analyzed via flow cytometry. Bar diagram shows the mean fluorescence intensity (MFI) of each surface marker normalized to the respective M0 control. Representative histograms of 3 independent experiments are shown (n = 5 donors, bar means ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 determined by two-way ANOVA).

Article Snippet: Labeled CD4+ T cells were plated in 48 well plates at 106 cells/mL in X-Vivo 15 (Lonza, #BE02-060F, Basel, Switzerland) and incubated for 3 days in the presence or absence of platelets derived from iPRF at a ratio of 1:50 as well as 10 μg/mL anti-GARP Ab (Origene) before undergoing flow cytometric analysis.

Techniques: Derivative Assay, Marker, Expressing, Cytometry, Control