Cytotoxicity Assay of Expanded Lymphocytes

The 14-day DC/PC-3/IL-2-expanded lymphocytes were reconstituted and cultured (37 °C, 5% CO₂) for 18–24 h in LM medium with or without IL-2 (80 IU/mL). The cells were pelleted, washed, and resuspended in fresh LM medium with or without IL-2 (80 IU/mL), and stimulated by coculturing with fluorescent TagFP635-PC-3 cells [39 (link)] at a ratio of 20:1 (lymphocytes:TagFP635-PC-3 cells). After 5-h coculturing, the cells were supplemented with Precision Count Beads (BioLegend), and the relative proportion of TagFP635-PC-3 cells to the Precision Count Beads determined by flow cytometry as described above. The cytotoxic activity was calculated as the difference between the relative proportion of TagFP635-PC-3 cells cocultured and not cocultured with DC/PC-3/IL-2-expanded lymphocytes.