Enzychrom nad nadh ratio assay kit

NAD+/NADH ratio was tested using EnzyChrom™ NAD+/NADH Ratio Assay Kit (Cat# E2ND-100, Bioassay Systems, CA, USA) (24 (link)). NAD+ and NADH extraction buffer were used respectively to resuspend cells for NAD+ and NADH determination. 10⁵ cells were collected for detection for each sample and washed by cold water. Homogenize samples were put in a 1.5 ml Eppindorf tube with either 100 ml NAD extraction buffer for NAD determination or 100 ml NADH extraction buffer for NADH determination. After heating at 60°C for 5 min, 20 μl of assay buffer was added into the extracts and 100 ml of the opposite extraction buffer was added to neutralize the extracts. Then made the calibration curve. Then the mixtures went for centrifugation at 13,000 rpm for 5e min. Supernatants were transferred to working reagent and the optical densities at 565 nm were read at 0 and 15 min. Absorbance values were used to calculate NAD+/NADH ratios according to the calibration curve.

NAD⁺/NADH ratio was tested using the EnzyChrom™ NAD⁺/NADH Ratio Assay Kit (E2ND-100, Bioassay Systems, CA, USA) [27 (link)]. Cells were suspended in NAD⁺ and NADH extraction buffers to conduct NAD⁺ and NADH assays. A total of 1 × 10⁵ cells were collected. The resulting homogenate was placed in a 1.5 ml Eppendorf tube, with 100 ml NAD extraction buffer added for NAD assay or 100 ml NADH extraction buffer added for NADH assay. Subsequently, 20 μl assay buffer was introduced to the extract, followed by the addition of 100 ml back extraction buffer to neutralize the extract. The mixture was then centrifuged, and the resulting supernatant was transferred to the working reagent. Optical density values at 565 nm were measured at both 0 and 15 min.