Lymphocyte Activation and PBMC Isolation

Single cell suspensions of lymphocytes were prepared from the spleen of sacrificed mice. Red blood cell lysis was performed with ACK Lysis Buffer (Quality Biologicals, MD, USA).(26 (link)) Human blood samples (buffy coats) were obtained from the National Institutes of Health Blood Research Services for peripheral blood mononuclear cell (PBMC) isolation and prepared as previously described.(27 (link)) Fresh PBMCs were isolated by Ficoll density gradient centrifugation (Biochrom, Berlin, Germany). Splenocyte or PBMC cell activation was performed with the Cell Activation Cocktail without Brefeldin A (BioLegend) according to the manufacturer’s protocol. Briefly, splenocytes or PBMCs were cultured in 1mL medium with 2μL of the Cell Activation Cocktail for 24 hours. Cells and supernatant were then transferred from the six well plate into an Eppendorf tube which was centrifuged and supernatant collected for coculture with tumor cells.