LRRC32/GARP Protein 6055-LR-050 from R&D Systems, a Bio-Techne Brand

Published Figures

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Biomedicines
December 05 2022

Fig 1: 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 Foxp3 (A), GARP (B), and proliferation (C) of CD4+ T cells was measured on day 3 of coincubation via flow cytometry. (D,E) After 6 days of coincubation, cells were stimulated with phorbol 12-myristate 13-acetate (PMA), ionomycin, and monesin for 5 h. IL-2 and IFN-γ production was quantified via intracellular flow cytometry. Representative dot plots of three independent experiments are shown. Graphs show CD4+ T cells cocultured with platelets normalized to untreated CD4+ T cells alone control (n = 3, means ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, and ns determined by one-way ANOVA).
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Biomedicines
December 05 2022

Fig 2: 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).
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Biomedicines
December 05 2022

Fig 3: 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).