Caspase glo 3 7 substrate

U2-OS cells were seeded in 96-well dishes at 10⁴ cells/well in DMEM supplemented with 10% FBS. After 48 h, cells were mock-treated or treated with staurosporine (STS) (0.5 μm, 6 h), doxorubicin (DOXO) (3 μm, 48 h), or cycloheximide (CHX) (20 μg/ml) and human tumor necrosis factor α (TNF-α) (10 ng/ml) for 16 h in DMEM with 2% FBS. Media used for mock treatments were supplemented with concentrations of the solvents used to dissolve drugs: DMSO for STS or DOXO and Milli-Q water for CHX and TNF-α. Caspase-3 and -7 activity was determined according to the manufacturer's instructions, using the Caspase-Glo 3/7 substrate (Promega). The resulting luminescence, which is proportional to caspase-3/7 activity, was measured using a luminometer (Omega).

Cells were seeded in a 384-well format in 20 μL medium. At the desired time point 10 μL Caspase Glo^® 3/7 Substrate (Promega) were added and plates vigorously shaken for 1 min. After 30 min incubation at RT in the dark luminescence was measured.

The cell viability (GF-AFC cleavage), cytotoxicity (bis-AAF-R110 cleavage) and apoptosis (caspase 3/7 activity) were measured simultaneously via the Apotox-Glo Triplex assay (Promega, Madison, WI). IRKD cells and control cells were plated (10⁴ cells/100 µl/well) in 96 well plates in complete growth media. Samples were assayed 0, 1, 2, 4 and 6 days after plating, as indicated, and were processed according to the manufacturer's instructions. The GF-AFC and bis-AAF-R110 substrates were added, and fluorescence was measured by the GloMax-Multi Detection System (Promega) (excitation 400 nm/emission 485 nm and 505 nm/520 nm, respectively) after a 30 min incubation at 37°C. Luminescence was detected after a 30 min incubation with the Caspase-Glo 3/7 Substrate (Promega). After the background for each assay was subtracted, the data were represented as the fold-change from baseline (day 0).