PBMCs isolation and serum collection

Approximately, 8–10 ml of peripheral venous blood were withdrawn from each participant for PBMCs isolation, while another 4 ml peripheral venous blood samples were collected from the participants for serum separation. Within 4–6 hrs of sample collection, PBMCs were isolated using the Ficoll-density gradient centrifugation method. Briefly, peripheral venous blood was diluted in an equal volume of wash mix (Roswell Park Memorial Institute (RPMI) 94%, fetal bovine serum (FBS) 5%, penicillin/ streptomycin 1%). Diluted blood, approximately 6 ml, was carefully layered on a 3 ml Ficoll reagent, followed by 30 min of centrifugation at 1000 rpm. The buffy layer containing the PBMCs was collected and washed twice with the wash mix. The PBMCs of each sample were cryopreserved and stored in a −80 °C freezer for later use as previously described [15,17]. Serum separation was done by centrifuging at 1500 rcf for 10 min, followed by the collection of the upper serum phase that was stored at −80 °C until further use.