3D spheroid generation and viability assay

3D spheroids of the U87 cell line and PDG cells were generated by seeding 5 × 10³/50 μl of cells to a low-adherent 96-well plate. On day 4, 2 × 10⁶ γδ T cells or culture medium was added to a final volume of 100 μl. After 24 hours of co-culture, the viability of the 3D spheroids was analyzed using CellTiter-Glo (Promega, Madison, WI) following the manufacturer’s protocol. Briefly, 100ul of CellTiter-Glo reagent was added to the wells after acclimating the spheroid and reagent to room temperature for 30 minutes. Wells were thoroughly mixed to lyse viable cells and luminescence was measured with Synergy H1 microplate reader (BioTek, VT). The luminescence of the 3D spheroids treated with γδ T cells was calculated as (luminescence of 3D spheroids with γδ T cells)-(luminescence of γδ T cells alone).