The induction of apoptosis by LTZ, TMZ and LTZ + TMZ combination as evident by caspase 3/7 activation was assessed in all four GBM lines, G76, BT142, G43 and G75. Cells were seeded at a density of 1.5 × 104 cells per well in a translucent walled, clear bottom, 96-well plate. Promega Caspase-Glo® 3/7 Assay system was employed to assess the Caspase 3/7 levels in cells treated with LTZ, TMZ, LTZ (40 nM) + TMZ (0–5 μM) and vehicle (0.1% DMSO) [31 (link)]. Cells were treated for 24 h after plating. Caspase-Glo® 3/7 3D substrate and Caspase-Glo® 3/7 3D buffer was mixed to form a 10 mL reaction mixture and 100 μL of the mixture was added to each well. The reaction was allowed to proceed at room temperature in the dark for 30 min as described in the Promega Caspase-Glo® 3/7 3D-Assay technical manual. The luminescence readings were carried out 24 h after the treatment using Synergy™ HTX Multi-Mode Microplate Reader. The experiment was repeated in presence of estradiol to assess the effect of estradiol on caspase induction in in four GBM lines used in this study. The data were normalized to control using Promega CellTiter- Glo® assay.