Preparation of PBMCs
As previously described (21), peripheral blood was collected in
S-Monovette K3 EDTA blood collection tubes (Sarstedt). 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.