Activated PBMC-Mediated Cytotoxicity Assay

After mixing interleukin-2 (IL-2) (20 IU ml-1) with PBMCs (STEMCELL Technologies, Vancouver, Canada), HNSCC cells (1 × 10⁶) were co-cultured with PBMCs (1 × 10⁷) for 4 days, followed by isolation of the activated PBMCs using Percoll density gradient centrifugation. The HNSCC cells FADU and Cal27 were labeled with CellTrace (Far Red, Invitrogen, Carlsbad, California, USA), and then cultured with activated PBMCs in a medium (STEMCELL Technologies, Vancouver, Canada) containing human CD3/CD28 tetramer antibody complex in polystyrene tube (FALCON, Canada) for 48 h at 37 °C with 5% CO₂ in air. Next, the cells were rinsed with PBS, fixed and permeabilized, followed by repair with Fix/Perm solution (BD Biosciences, Franklin Lakes, NJ, USA). The cells were then stained with V450-cleaved caspase 3 antibody (560,627, dilution ratio of 1: 50, BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using BD FACS Canto II (BD Immunocytometry Systems, BD Biosciences, Franklin Lakes, NJ, USA). The HNSCC cells were isolated from the HNSCC/PBMC cell mixture by gating for the CellTrace⁺. Lastly, the activated PBMC-mediated killer HNSCC cells were analyzed by detecting the signal of the V450-cleaved caspase 3 [16 (link)].