MTT Cell Viability Assays

MTT assays were performed using the Vybrant MTT Cell Proliferation Assay kit (Invitrogen) according to the manufacturer’s instructions. Briefly, 200 μL of cells at a density of 5×10⁴ cells/mL were plated in wells of a 96-well plate and incubated for 24 hours. After 24 hours, cell solution was removed and replaced with 200 μL of media (control) and concentrations of ATP at 200 μM, 1 mM, or 5 mM and allowed to incubate for 48 hours. ATP-containing solutions were pH’d to physiological pH and filter-sterilized prior to use. Following incubation, media was removed and replaced with MTT reagent. Cells were then left to incubate for another 4 hours after which all of the solution was removed and replaced with 100 μl DMSO to dissolve the purple formazan crystals created by reduction of the MTT reagent during the incubation period. After 10 minutes, plates were shaken for 1 minute in a BioRad Benchmark Plus microplate reader and analyzed at a wavelength of 570 nm. Co-treatment toxicity studies were done using the above protocol for treatments with 100 μM o-ATP + media (as vehicle control), 100 μM o-ATP + 200 μM ATP, and 100 μM o-ATP + 1 mM ATP.