Glomax 96 well plate reader

Cell viability was assessed by harvesting A673 cells or CRISPR/Cas9 modified cells and seeding in a 96-well white, opaque, tissue-culture treated plate (Corning) at a density of 5,000 to 8,000 cells per well in 200 µL of media. After adhering overnight, cells were treated with increasing concentrations of SP-2509 (50 µL, 0.01–30 µmol/L final concentration), or vehicle control (0.1% DMSO) for 72 hours, and each condition was repeated in triplicate. For ETC inhibitor studies, these inhibitors were added during the SP-2509 treatment step at a final concentration of 0.5 µmol/L for antimycin A and oligomycin A, and 0.05 µmol/L for rotenone. After 72 hours, 170 µL of media were removed from each well, and 80 µL of CellTiter-Glo (Promega) reagent was added to each well. Plates were agitated at 700 rpm for 2 minutes and allowed to incubate for 10 minutes shielded from ambient light. Luminescence was measured on a GloMax 96-well plate reader (Promega) and the cell viability was calculated relative to the vehicle treated wells. IC₅₀ values were determined by using non-linear regression software GraphPad Prism 8.0 and fitting the data to log(SP-2509) versus response 4-parameter variable slope.

Enzyme activity was determined with ADP-Glo-Kinase assay according to the manufacturer’s protocol (Promega). Briefly, FX-9 (0.1–5 μM), CX-4945 (0.1–5 μM) or DMSO was incubated with 50 ng recombinant human CK2α1 (Promega), 2.5 μg CK2α1 substrate (Promega), 10 μM ATP for 60 min at room temperature. The kinase reaction was terminated by ADP-Glo Reagent to deplete remaining ATP. In a finally step a conversion of ADP to ATP was performed with kinase detection reagent to generate luminescence using a luciferase/luciferin reaction. Luminescence was measured with the Glomax 96 well plate reader (Promega). The emitted light correlates with the amount of ADP generated in the kinase assay which is indicative of kinase activity.