Multiparameter Flow Cytometry of PBMCs

0.5-2x10⁶ PBMCs were stained for 15 min at 4°C with different combinations of antibodies (Table S2) and washed before acquisition. 4,6-Diamidine-2-Phenylindole (DAPI) (Molecular Probes, Eugene, USA) or LIVE/DEAD Fixable Blue Dead Cell Stain Kit (ThermoFisher Scientific, Waltham, USA) was used to identify dead cells according to the manufacturer’s protocol. Cells were acquired on a FACS Canto II or LSR Fortessa X-20 flow cytometer (BD Biosciences, Heidelberg, Germany) (Figure S1A) (19 (link)). For quality control, CS&T Beads (BD Biosciences), SHPERO Calibration Particles (BD Biosciences) and in some experiments, application settings have been used to obtain reproducible median fluorescence intensities (MFIs). The isotype control of BTLA was measured in a fluorescence minus one (FMO) control approach.