ATP Synthesis Measurement in Cells

ATP synthesis was evaluated using a commercially available luciferase-luciferin system (ATPlite, Perkin Elmer, Waltham, MA). The day before the experiment, cells were plated (5,000 cells/well) in 96 multiwell plates. The day after, cells were first washed with Tyrode’s solution containing (in mM): NaCl 137, KCl 2.7, MgCl₂ 1, CaCl₂ 1.8, NaH₂PO₄ 0.2, NaHCO₃ 10, glucose 5.5 mM, pH 7.4 and then exposed to different glutamate concentrations (0.5 and 1 mM) in the same Tyrode’s solution for 1 h at 37 °C¹⁷. When ATP content was evaluated after H/R, glutamate and the specific pharmacological tools were added at the beginning of the reoxygenation phase and maintained for 1 h. After the incubation period, ATP levels were analyzed with a luminescence counter (Victor Multilabel Counter, Perkin Elmer) and normalized to the respective protein content^{17 (link),18 (link)}.