Blood Sample Collection and PBMC Isolation

We obtained approximately 30 ml of blood in S-Monovette K3 EDTA tubes and 7 ml in S-Monovette Serum-Gel tubes (Sarstedt). All samples were collected in the morning (8:00 a.m.). PBMCs were then isolated using a Ficoll–Hypaque gradient as described (17 (link)), aliquoted in RPMI containing 10% DMSO and 25% FCS at 1 × 10⁷ cells/ml, gradually cooled down to −80°C in a Mr. Frosty for 18 h and stored in liquid nitrogen until assayed. A small amount of whole blood (50 µl) was used for total and differential leukocyte counts using a Coulter Ac·T Diff hematology analyzer (Beckman Coulter). All other assays were performed using cryopreserved PBMCs or serum with one subject from each group run in parallel to control systematic variation in reagents used.