Fixable yellow dead cell stain kit

The Treg flow cytometry panel was inspired by a clinical Tregs workshop (21 (link)). Blood was sampled in heparin-containing tubes and peripheral mononuclear cells (PBMCs) were isolated by Ficoll density gradient separation following standard procedures before freezing down at -150°C in AB serum with 10% DMSO. For flow analysis, thawed PBMCs from 5 patients and 8 controls (see Table 1) were added two microliter of Fc block for 15 min before addition of the extracellular antibodies anti-CTLA4 BV421 (BN13, Biolegend, San Diego, CA), anti-CD39 PE (ebioA1, Invitrogen, Carlsbad, California, USA) and anti-CD3 V500 (UCHT1); anti-CD4 Alexa Fluor 700 (RPA-T4); anti-CD8 PerCP-Cy5.5 (SKI); anti-CD25 PE-Cy7 (2A3); anti-CD45RA APC-H7 (HI100) and anti-CD31 BV650 (L133.1) from BD (Franklin Lakes, New Jersey, USA). The cells were resuspended in 1 mL PBS and live/dead Fixable Yellow Dead Cell stain kit (Invitrogen) was added in a 1:1000 dilution. Staining of intracellular markers [anti-FOXP3 PE-CF594 (259D/C7, BD), anti-HELIOS APC (22F6, Biolegend), and anti-Ki-67 (20Raj1, Invitrogen)] was subsequently performed using the eBioscience Anti-human FOXP3 Staining Set as explained by the manufacturer with the change that the cells were fixed overnight. Acquisition was executed by a LSR Fortessa flow cytometer and data was analyzed by FlowJo version 10.4.