Following patient recruitment, venesection was performed at induction of anesthesia and 24 h after surgery and 8 mL of blood drawn into a cell preparation tube with sodium heparin (CPT™) vacutainer and a further 5 mL drawn into a serum tube [Beckton Dickinson (BD), Wokingham, UK]. Samples were processed within 1 h of venesection. CPT™ vacutainers were centrifuged at 1,500 g for 15 min at room temperature and the peripheral blood mononuclear cell (PBMC) layer extracted, washed twice in 2 mL of phosphate-buffered saline (PBS) before being resuspended in freezing media (fetal bovine serum [FBS; Gibco, Thermo Fisher (TF), Cambridge, UK] with 10% dimethyl sulfoxide (Sigma) and frozen to −80°C in a Mr Frosty™ and transferred to liquid nitrogen within 48 h for long-term storage.
Samples were analyzed in batches. Frozen PBMCs were defrosted by resuspension in RPMI Glutamax medium (Gibco) with 10% FBS (TF), washed once in media, counted (Countess 3 Automated cell counter, TF), and diluted to a concentration of 1 × 106/mL. PBMCs were either labeled with antibodies for flow cytometry or cultured for in vitro stimulation.
Samples were analyzed in batches. Frozen PBMCs were defrosted by resuspension in RPMI Glutamax medium (Gibco) with 10% FBS (TF), washed once in media, counted (Countess 3 Automated cell counter, TF), and diluted to a concentration of 1 × 106/mL. PBMCs were either labeled with antibodies for flow cytometry or cultured for in vitro stimulation.