Percp cy5.5 conjugated anti cd45ra clone hi100

PBMCs were incubated in the AIM-V medium with β-mercaptoethanol at 37°C, 5% CO₂ for 7 days, with or without 5 μg/ml recombinant GAD₆₅. Thereafter, cells were washed in PBS containing 0.1% BSA and subsequently stained with Alexa 700-conjugated anti-CD3 (clone UCHT1, BD Biosciences), Pacific Blue-conjugated anti-CD4 (clone RPA-T4, BD Biosciences), allophycocyanin- (APC-) H7-conjugated anti-CD8 (clone SK1, BD Biosciences), PerCP-Cy5.5-conjugated anti-CD45RA (clone HI100, BD Biosciences), phycoerythrin- (PE-) conjugated anti-CCR7 (clone G043H7, BioLegend), FITC-conjugated anti-CD127 (clone eBioRDR5, eBioscience), and PE-Cy7-conjugated anti-CD25 (clone BC96, eBioscience). Then, cells were fixed and permeabilized using the FOXP3-staining buffer set (eBioscience), according to the manufacturer's instructions. Cells were then stained with APC-conjugated anti-FOXP3 (clone PCH101, eBioscience) and acquired on a FACS Aria III (Becton Dickinson) running FACS Diva v8 software (Becton Dickinson). Data were analyzed using Kaluza v1.3 (Beckman Coulter).

Blood was collected in EDTA K2 tubes. PBMCs were isolated from 10 mL of whole blood using Ficoll-Paque Plus (GE Healthcare, Aulnay-sous-bois, France), cultivated on 96-well plate at 1.5 × 10⁶ cells/mL for 24 h in filtered complete media (RPMI 1640 with 10% fetal calf serum, penicillin and streptomycin). The day after, PBMCs were stained and sorted using FACS ARIA III (BD biosciences). To isolate CD4⁺CD25⁻ cells, we used FITC-conjugated anti-CD4 (clone RPA-T4, BD Pharmingen) and PE-conjugated anti-CD25 (clone BC96, ebioscience, Thermofisher, USA) antibodies. To sort naïve CD4⁺CD45RA⁺CD62L⁺ cells, we used APC eFluor 780-conjugated anti-CD4 (RPA-T4, ebioscience), FITC-conjugated anti-CD62L (clone DREG-56, BD Pharmingen), and PerCP Cy5.5-conjugated anti-CD45RA (clone HI100 BD Pharmingen) antibodies. Dead cells were excluded using DAPI staining. Gating strategies are shown Figure S1 in Supplementary Material.