2.3. ATPlite luminescence assay

ATP generation in passage 1 hAF cells treated with different concentrations of polyP‐22 was measured using ATPlite Luminescence Assay System (catalog number 6016941, PerkinElmer) following manufacturer's instructions. hAF cells were seeded onto a 96‐well plate at a density of 2000 cells/well and cultured for 24 hours in 100 μL of Ham's F‐12 media supplemented with 5% FBS and 1% P/S. Following day, the culture media was replaced with 100 μL fresh culture media supplemented with different concentrations of polyP‐22 (0, 0.5, and 1.0 mM) and cultured for up to 72 hours.

At each time point (1, 3, 6, 12, 24, 48, and 72 hours.), culture media was removed and 50 μL of mammalian cell lysis solution (kit component) was added to the culture wells and the culture plate shaken for 5 minutes. Then 50 μL substrate solution was added to the wells and the plate was shaken again for 5 minutes. The plate was dark adapted in the luminescence plate reader (VICTOR III Light luminescence plate reader) and luminescence was read. ³³ The total DNA content (ng) of each sample well was measured using a Quant‐iT PicoGreen ds DNA Assay Kit (Invitrogen, Eugene, Oregon) and generated ATP (nM) amount was expressed normalized to total DNA content (nM/ng).