2.4. ATP content

Cellular adenosine triphosphate (ATP) content was determined using the ATP Determination Kit (#A22066; Molecular Probes, Eugene, OR) following the manufacturer’s instructions. Briefly, on DIV 7, hippocampal neurons were treated with HU (5 μM). Approximately 24 hours later, H₂O₂ peroxide was added (15 μM or 35 μM) for one hour. All medium was removed, 11 μl boiling water was added to each well, and 110 μl of ATP reaction mixture was added and mixed with the lysed cells. ATP content was determined by measuring luciferase activity compared to a standard curve. The concentration of ATP per treatment condition was normalized relative to the untreated control neurons. N ≥ 3 independent experiments.