Nadp nadph ratiometric kit

RCECs were seeded onto gelatin-coated six-well plates, allowed to adhere overnight and exposed to experimental conditions. After treatment of 10 μM NR1 for 72 h, cells (1×10⁶ per sample) were lysed in 100 μL of lysis buffer to perform the NAD⁺/NADH assays. The NAD⁺ and NADH levels were measured using the NAD⁺/NADH Assay Kit (AAT Bioquest) according to the manufacturer’s instructions. The intensity of the reduced product color, measured at 460 nm, is proportionate to the NAD⁺ concentration in the sample. The standards supplied with the kits were used to prepare the calibration curves needed to calculate the NAD⁺/NADH ratio. NADP⁺/NADPH measurements were performed using the NADP⁺/NADPH Ratiometric Kit (Promega, Madison, WI, USA). Cell extracts (5×10⁴ per sample) were treated with the respective NADP⁺/NADPH extraction buffer, and chemiluminescence was measured on a 2300 EnSpire Multimode Plate Reader (PerkinElmer, Boston, MA, USA).