96 well u bottom culture plates

For T-cell stimulation, 96-well U-bottom culture plates (Corning, NY, USA) were coated overnight at 4°C with 1μg/mL α-CD3 antibodies (ThermoFisher) and washed twice with 1x PBS. 2x10⁶ thawed PBMCs in RP-10 media were then stimulated with 2μg/mL soluble α-CD28 antibodies (BD Biosciences) for 6 hours. For T-cell activation by CEF (cytomegalovirus, Epstein-Barr virus, and influenza virus) combo peptides, thawed PBMCs in RP-10 media were activated in 96-well U-bottom culture plates (Corning) with 1μg/mL of CEF combo peptides (NIH AIDS Reagent Program, MD, USA) for 6 hours. α-CD3/CD28 stimulation is MHC-independent, allowing for a stimulation signal for bulk T-cells in PBMCs while CEF peptides are a stimulation for a viral specific memory CD8 T-cells [30 (link), 31 (link)].