Anti human cd3 pe cy7 clone ucht1

The antibody concentrations are indicated in ng or test (as indicated by the manufacturers if the real concentration for the product is not available) as follows. Anti-human CD8 Brilliant Violet (BV) 510 (clone RPA-T8, used at 0.1 test/50 μL), anti-human CD3 PE-Cy7 (clone UCHT1, used at 0.1 test/50 μL) and anti-human CD34 APC (clone 8G12, used 50 ng/50 μL) were purchased from BD Biosciences (NSW, Australia). Anti-human CD4 PE-Cy7 (clone OKT4, used at 150 ng/50 μL), anti-human CD4 PerCP-Cy5.5 (clone OKT4, used at 25 ng/50 μL) and anti-human CXCR4 BV421 (clone 12G5, 50 ng/50 μL) were purchased from BioLegend (CA, USA). Anti-human CD3 ECD (clone UCHT1, used at 0.05 test/50 μL) and anti-HIV-1 p24 PE (clone FH190-1-1, also known as KC57 RD1, used at 0.1 test/50 μL) were purchased from Beckman Coulter (NSW, Australia). Anti-human CD7 FITC (clone CD7-6B7, used at 50 ng/50 μL) was purchased from CALTAG Laboratories (CA, USA). Optimal concentrations per test were determined by flow cytometry prior to the experiments.

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient centrifugation. After isolation, PBMCs were stained for 45 min at 4°C in PBS 0.1% bovine serum albumin with the following antibodies: anti-human CD3-PE-Cy7 (clone UCHT1, BD Biosciences), anti-human CD4-FITC (clone 13B8.2, Beckman Coulter), anti-human CD8α-BV421 (clone RPA-T8, BD), anti-human CCR6-PE (clone G034E3, Biolegend), and anti-human CXCR6-APC (clone K041E5, Biolegend). Fluorescence was measured on a BD LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo or DIVA softwares. DP8α Tregs (CD3⁺CD4⁺CD8α^loCCR6⁺CXCR6⁺ cells) were then quantified among total CD3⁺ T cells, as described previously (Godefroy et al., 2018 (link)). The gating strategy is shown in Supplementary Figure S2.