2.6. ATP measurement

For intracellular ATP measurement, cells were lysed using RIPA buffer. Cell lysate was diluted 1:100 in ultrapure water and CellTiter-Glo from Promega (Cat#: G7570) was used to measure ATP levels following manufacturer’s instructions. Briefly, 50 μL of cell lysate dilution was mixed with 50 μL of CellTiter-Glo reagent and incubated for 10 min at room temperature. Bioluminescence was measured using a GloMax 20/20 luminometer. Values obtained were compared to a standard curve to determine ATP concentration. ATP values were normalized to protein levels for each sample.

For extracellular ATP measurements, supernatant from each sample was collected and incubated at 95°C in a heat block for 5 min to inactivate any ATPases present. Supernatants were mixed with CellTiter-Glo reagent; measurements were performed the same way as for intracellular ATP measurements described above.