Celltiter glotm luminescent cell viability assay kit

ATP levels in viable cells were quantified using CellTiter-GloTM Luminescent Cell Viability assay kit (Promega) according to the manufacturer's instructions. Lyophilized enzyme/substrate mixtures (100 μl) were transferred to opaque 96-well microplates containing cell lysates. The microplates were then incubated at room temperature for 10 min to stabilize luminescence signals, which were then measured using a Tecan GENios Microplate Reader.

A total number of 12,000 cells per well were seeded in a 96-well plate and then treated with different concentration of drugs (SNS-032 and AZD4573, obtained from Selleck, Houston, TX, United States) for 72 h. Cell viability was evaluated using CTG (Promega CellTiter-Glo^TM Luminescent Cell Viability Assay Kit) according to the manufacturer’s protocol. The absorbance optical density of 405 nm was recorded using a microplate reader (Synerge2; BioTek Instruments, Winooski, VT, United States), and the half maximal inhibitory concentration (IC50) was calculated using GraphPad Prism.

The CellTiter-Glo^TM luminescent cell viability assay Kit (Promega, Madison, WI) was used to evaluate the intracellular ATP content. Cells were seeded in 96-well plates. After 24 hours of attachment to the bottom of the plates, 50 μl of the CellTiter-Glo reagent was added directly into each well for a 10-minute incubation. The plate was read by an EnSpire^TM Multimode Plate Reader (PerkinElmer, Waltham, MA, USA) after incubation to monitor the luminescence signal generated by the luciferase-catalyzed reaction of luciferin and ATP. Assays were performed in triplicate at least twice. ATP content was then calculated by comparing the luminescence levels of RCC4-VHL cells with that of RCC4-EV cells, which was defined as 100%. Data were expressed as mean ± standard deviation (SD).