Cytotoxicity Assay for OVCAR3 Cells

We plated 10,000 OVCAR3 cells per well in a 96-well plate and, after 18 hours, added primary PBMCs. After a 4-hour coincubation, we stained the cells with 7AAD (Invitrogen), annexin V (BioLegend), and anti–human CD45 (304002, BioLegend), then performed a flow cytometry–based cytotoxicity assay as described previously (7 (link)). We stably transfected K562 and K562-FSHR with firefly luciferase. We plated 20,000 K562 and K562-FSHR cells expressing luciferase in a 96-well plate and coincubated them for 5 hours with PBMCs. After the incubation, we lysed the cells and measured luciferase expression using CytoTox Glo (Promega) as previously described (32 (link)). Cytotoxicity was calculated as (maximum viability control – individual well)/(maximum viability control – maximum death control) × 100 as a percentage or relative to the control (PBMCs with mouse IgG2a isotype control C1.18.4).