2.7.1 ATP quantification
ATP was measured using CellTiter-Glo Luminescent Cell Viability Assay (Promega, Portugal). The reaction is catalyzed by the enzyme luciferase obtained from the firefly (Photinus pyralis ) to quantify ATP. The MgATP2- converts the luciferin into a form capable of being catalytically oxidized by the luciferase in a high quantum yield chemiluminescent reaction.
For that, either cell cultured in cell culture flasks or encapsulated in eSF hydrogels were placed in 96-well plates. Then, the analysis was performed following the manufacturer’s instructions. Briefly, CellTiter-Glo Reagent agent was added (150 µL) to each well of the 96-well plate and incubated for 10 min at room temperature to lysate cells. Then, the cell lysate (100 µL) was transferred to the wells of a 96-well white opaque microtiter plate (in triplicate). Results were obtained using the luminometer (Perkin-Elmer, USA). The signal intensity of the samples was measured. Results were obtained as integral relative light units (RLUs). An ATP calibration curve was generated using ATP solutions ranging from 5 to 40,000 cells, and luminescence readings from experimental samples were then extrapolated.