Measurement of intracellular ATP

Cellular ATP levels were determined using the ATP-based CellTiter-Glo luminescent Cell Viability kit (Promega, Madison, USA) according to the manufacturer’s instructions with the following modifications. Briefly, Cells were plated in triplicate in 96-well plates to allow for attachment overnight, and then the culture was switched to glutamine-free medium or L-asparaginase (L-ASP) was added to the culture for different times to deplete glutamine. The cell samples were then mixed with equal volume of the single-step reagent provided with the ATP-based CellTiter-Glo kit and rocked for 2 min followed by incubation at room temperature for 15 min. Then luminescence levels were measured using a luminescent plate reader (Thermo Fisher Varioskan Flash; Waltham, MA).