K562 cells were plated at a density of 10,000 cells per well in 50 µL volumes of RPMI1640 plus 10% fetal bovine serum and allowed to attach for two hours. Saurosporine and ionomycin were twofold serially diluted and added to wells in 50 µL volumes. Plates were incubated at 37°C in 5% CO2 for 4–48 hours. MultiTox-Fluor™ Reagent was prepared as 10 µL of each substrate in 1 mL assay buffer, and 10 µL was used per well. The plate was mixed and incubated for 30 minutes at 37°C. Fluorescence as measures of viability and cytotoxicity was read at two wavelengths on a Tecan Safire2 reader. Caspase-Glo 3/7 Reagent was then added in an additional 100 µL volume, and luminescence was measured after 30 minutes at room temperature on a GloMax®-Multi Plate Reader.
1. Sterilely dispense 50 µL K562 cells in RPMI1640 plus 10% FBS culture medium into clear-bottom white tissue culture treated 96-well assay plates at a density of 10,000 cells per well.
2. Manually dispense 200 µL test compounds at double the highest desired concentration into column 12 wells of a second 96-well plate. Backfill (100 µL medium plus carrier) and generate serial dilutions of compounds across the plate by transferring 100 µL to the neighboring well, mixing, and repeating. Transfer 50 µL prepared compounds to respective wells of the assay plate containing cells. Shake the plate briefly on an orbital shaker and place at 37°C and incubate for the desired test exposure period (4, 8, 24, or 48 hours).
3. After the desired treatment time, dispense 10 µL MultiTox-Fluor Reagent to all wells, shake briefly, incubate at 37°C for 30 minutes, and measure resulting fluorescence using a fluorometer (live-cell fluorescence 400Ex/505Em; dead-cell fluorescence 485Ex/520Em). The MultiTox-Fluor™ Reagent was prepared by adding 10 µL of each substrate to 1 mL of assay buffer for this triplex protocol, see Promega Technical Bulletin #TB348 for details.
4. Dispense 100 µL Caspase-Glo® 3/7 Reagent to each well of the assay plate, orbitally shake briefly to lyse the cells, incubate at room temperature for 30 minutes, and measure luminescence using the GloMax®-Multi luminometer.
5. Repeat Steps 3 and 4 after each respective treatment time.