Pennicillin streptomycin

We collected blood in Vacutainer tubes (BD, Franklin Lakes, NJ) and transported immediately to the lab. We then centrifuged the whole blood at 2,000 RPM for 10 minutes at 24°C to separate cells and plasma, then collected 1 mL aliquots of plasma and stored them at −80°C. We stored biopsies in RPMI 1640 medium (GE Healthcare Life Sciences, Pittsburgh, PA) upon collection and immediately transported them to the laboratory. We stored two biopsies at −80°C and one in RNALater solution as noted above for DNA extraction and RNAseq analysis, respectively. We placed the remaining biopsies into 50mL RPMI medium supplemented with 1X Pennicillin/Streptomycin (GE Healthcare Life Sciences), 40 μg/mL Liberase TL (Sigma Aldrich), and 4 μg/mL DNase (Sigma Aldrich) and vigorously stirred at 37°C for one hour. We then manually dissociated the digested biopsy tissue into a single cell suspension by grinding them across a sterile 40μm mesh filter. We then centrifuged the cells at 1,900 RPM for 6 minutes at 4°C, suspended them in 8mL RPMI 1640 supplemented with 10% FBS, and 1X Pennicillin/Streptomycin (R10), and divided this suspension evenly between two 5mL round-bottom tubes for flow cytometry analysis.