Cytotoxicity Assay of Prostate Tumor Cells

Tumor cells were either isolated from fresh prostate tumor tissue procured from LF’s lab at UCSF (San Francisco, California, USA) or received as frozen dissociated aliquots along with donor matched PBMCs from Discovery Life Sciences (Newtown, Pennsylvania, USA). The fresh prostate tissue was dissociated as previously described.31 (link) For the cytotoxicity assay, exogenous human PBMCs were co-cultured with dissociated tumor cells at an E:T of 1:2 for 24 hours at 37°C, 8% CO₂. The death of PSMA⁺ cells was analyzed by flow cytometry on the BD FACSCelesta. An anti-CD45 antibody (BioLegend) was used to distinguish hematopoietic cells, and a non-epitope competing anti-PSMA antibody (clone LNI17, BioLegend) was used to identify PSMA⁺ cells. The percentage of PSMA⁺ live cells was determined using eBioscience Fixable Viability Dye eFluor 780 (Invitrogen, Carlsbad, California, USA). Culture supernatant from each well was used to measure IL-2, IFNγ, and TNFα by MSD.