PBMC Isolation and Cryopreservation Protocol

5–10mls of peripheral blood was taken through venous puncture and stored in acid/citrate/dextrose tubes. PBMCs were isolated by density centrifugation in Ficoll histopaque 1077 using SepMate PBMC isolation tube (StemCell tech, Cat#: 85415) according to the manufacturer’s protocol. After the cells were washed in buffer (0.1 BSA, 2mM EDTA, 1X PBS), residual red blood cells were removed using red blood cells lysis buffer (EBioscience, cat#: 00–4300-54). The cells were then frozen at a density of 1x10⁶/ml in freezing media containing (90%FBS Heat Inactivated (HI), 10% DMSO) and stored at −80°C in a Mr. Frosty apparatus (Nalgene) for 24 hours before being transferred to long-term storage in liquid N2 until use as previously described ^{[22 (link)]}.