Human PD-1 QuicKit ELISA

Human peripheral blood mononuclear cells (PBMCs) were cultured at 3 x 106 cells/mL in RPMI supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin and were either left untreated or treated with 10 ug/mL PHA-L and 20 ng/mL of recombinant human (rh) IL-2 for 5 days. After 5 days, cells were then treated with 10 ng/mL PMA and 500 ng/mL Ionomycin for an additional 24 hours before harvesting conditioned media. CD4+ T cells were isolated from PBMCs using MagCellect™ Human CD4+ T Cell Isolation Kit (R&D Systems®, Catalog # MAGH102). For Cloudz™ stimulation, CD4+ T cells were cultured at 5 x 105 /mL using ExCellerate™ Human T Cell Expansion Media, Xeno-Free (R&D Systems, Catalog # CCM030) for the duration. T cells were treated with 25 μL of Cloudz CD3/28 particles per mL of culture media for 5 days. Cell conditioned media was collected by centrifugation and stored at ≤ -20 °C until assayed. After centrifugation, the remaining cells and Cloudz particles were resuspended with 1X Release Buffer. After dissolution of Cloudz particles, cells were lysed as indicated on the next page.