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.