Flow Cytometry-based Isolation of T-Cell Subsets

PBMC were isolated from heparinised venous blood or buffy packs by using lymphoprep© (Stem Cell Technologies) density gradients. Resulting PBMC were frozen in liquid nitrogen and thawed to be used for subsequent experiments. Cells were sorted on an ARIA III Fusion Cell Sorter (BD Bioscience) at the University of Birmingham or FlowCore, Monash University. PBMC and purified T-cells were cultured in RPMI-1640 medium (Invitrogen) supplemented with 2 mM L-glutamine, 1% sodium pyruvate, 50 μg/mL penicillin/streptomycin (Invitrogen) and 10% fetal calf serum (Sigma). In total, Vδ1⁺ T_effector (CD27^lo/neg) populations were sorted from 6 healthy donors, and Vδ1⁺ T_naïve (CD27^hi) from 3, CD8⁺ T_naïve from 7, and CD8⁺ T_EMRA populations were obtained from 9, with comparator Vδ2⁺ populations obtained from 2 individuals (Davey et al., 2017 (link)).