Isolation and Cryopreservation of PBMCs

As previously described, peripheral blood was collected in S-Monovette K3 EDTA blood collection tubes (Sarstedt) (22 (link)). Collected blood was prediluted in PBS/BSA (Gibco) at a 1:1 ratio and underlaid with 15 mL of Ficoll-Paque Plus (GE Healthcare). Tubes were centrifuged at 800g for 20 min at room temperature. Isolated PBMCs were washed twice with PBS/BSA and stored at -80°C until use. The cryopreserved PBMCs were thawed by incubating cryovials 2-3 minutes at 37°C in bead bath, washed twice in 37°C RPMI 1640 media (Life Technologies) supplemented with 1% penicillin-streptomycin-glutamine (Sigma-Aldrich), and 10% fetal calf serum (FCS) (PAN-Biotech) medium, and incubated overnight at 37°C.