Daan Pieren

124 Chapter 5 PBMC isolation and flow cytometry Peripheral Blood Mononuclear cells (PBMCs) were isolated by density gradient (Ficoll-Hypaque, Amersham Biosciences) from heparinized blood or buffy coats and stored at -135 °C in 10% dimethyl sulfoxide (DMSO, Sigma Aldrich) and 10% fetal calf serum (FCS) until further use. For ex vivo flow cytometric analyses, frozen PBMCs were thawed at 37°C and were transferred to RPMI-1640 medium (GIBCO, Thermo Fisher Scientific) supplemented with 10% FCS and Penicillin- Streptomycin-Glutamine (P/S/G) and washed twice. Thawed cells were then rested in medium for 30 minutes at room temperature after which the number of viable cells was determined on a Coulter Counter (Beckman). For ex vivo analyses on all healthy individuals, 4*10 5 PBMCs of each individual were labeled for surface markers at 4°C for 30 minutes with saturating concentrations of antibodies targeting: CD3, CD4, CD8, CD27, CD45RA, CD122, CD226, KIR2D, KIR3DL1, NKG2A, and TIGIT ( listed in Supplementary Table 4 ) in FACS buffer (1x PBS +0.5% BSA +2mM EDTA). A Fixable Viability Stain 780 (BD Horizon) was added to the labeling to identify viable cells. Cells were then fixed and permeabilized with buffers for subsequent intracellular labeling (eBioscience) at 4°C for 30 minutes with an antibody targeting Helios ( Supplementary Table 4 ). For ex vivo analyses on symptomatic individuals and their asymptomatic/ healthy controls, 2-6*10 6 PBMCs were first labeled with an HLA-A2 GILG- dextramer (A*0201/GILGFVFTL dextramer, FITC-conjugated, Immundex) for 20 minutes at room temperature. Cells were then labeled for surface markers at 4 °C for 30 minutes with saturating concentrations of antibodies targeting: CD3, CD4, CD8, CD27, CD45RA, CD69, KIR2D, KIR3DL1, NKG2A, and TIGIT in FACS buffer, before intracellular labeling of Helios as described above ( listed in Supplementary Table 4 ). A Fixable Viability Stain 780 (BD Horizon) was used to identify viable cells. Samples were measured on an LSRFortessa™ X-20 (BD Biosciences) and data were analyzed using FlowJo software (v10.6.1, TreeStar). T-cell culture assays Cell sorting for T-cell cultures For all culture assays we used PBMC that were collected from healthy blood donors (Sanquin Blood Supply Foundation) and thawed upon storage at -135 degrees Celsius. The number of viable cells in thawed PBMC samples was determined manually using trypan blue staining and Bürker-Türk. For T-cell suppression and T-cell proliferation/activation assays, (part of) the cells were washed with 1xPBS and labelled with 0.5 µ M CellTrace TM Violet (Invitrogen) in 1x PBS per milliliter of cell suspension (10 6 cells/mL) for 20 minutes at 37 °C to