Colorimetric Assay for Cellular NAD+ Levels

Cellular NAD⁺ levels were measured using an NAD⁺/NADH Colorimetric Assay Kit (ab65348, Abcam, Cambridge, UK). PBMCs (5 × 10⁶ cells) were incubated in nonadherent tubes in a 5% CO₂ incubator at 37 °C under unstimulated conditions and with different concentrations of olaparib (1, 10, and 100 µM) for 4 h. H₂O₂ (Sigma-Aldrich, 250 µM) was added for the final 2 h. (The concentration of the oxidant was selected based on pilot studies; we aimed to achieve an approximately 50% decrease in cellular NAD⁺ and ATP levels). After washing, cell lysis was performed using protease inhibitor extraction buffer according to the manufacturer’s instructions. The samples were centrifuged at 16,000× g for 5 min at 4 °C. The supernatant was collected and stored on ice. Six microliters of the sample was removed for subsequent protein quantification. The remainder was transferred to a 10 kDa Spin Column supported on a microtube and centrifuged at 10,000× g for 30 min at 4 °C to remove proteins and enzymes that could degrade NAD⁺. The filtrate was subjected to rapid freezing in liquid nitrogen and stored at −80 °C. Protein quantification was performed by the colorimetric method using a bovine serum albumin standard curve at 550 nm using a Multiskan Ex device (Thermo Fisher, Waltham, MA, USA). NAD⁺ was measured using a colorimetric method as described in [21 (link)].