Enzychrom nad nadh assay kit

The levels of NAD and NADH were measured using EnzyChrom NAD/NADH Assay Kit (E2ND-100; BioAssay Systems, Hayward, CA). IMR90 cells were grown in the presence or absence of 100 nM MB for 0–24 h, harvested, and the cells were collected in ice-cold PBS. About 10⁵ cells were used to assay NAD and NADH as described by the manufacturer. Briefly, two separate sets of 10⁵ cells were used. One set was extracted by 100 µl NAD-extraction buffer for NAD determination while the other set was extracted by NADH-extraction buffer for NADH determination. Both extracts were heated for 5 min at 60 °C. The level of NAD and NADH was assayed using enzymatic recycling of MTT followed by measuring the change in absorbance at 565 nm at the time points 0 and 15 min using Molecular Devices Spectra MaxM5. In conjunction with the unknown samples a standard curve was similarly prepared as described by the manufacturer. NAD and NADH concentrations were calculated using the difference in absorbance at 0 and 15 min and the standard curve. The concentration of NAD and NADH were used to calculate the ratio NAD/NADH at each time point.

NAD/NADH ratio was measured in lung tissue using EnzyChrom™ NAD/NADH Assay kit (Cat # E2ND-100, BioAssay Systems, USA) as per manufacturer's instructions. Briefly, two tissue samples from same animal, weighing 20–25 mg each, were taken and homogenized in 100 μl of NAD or NADH extraction buffer, respectively. The extracts were heated for 5 min at 60 °C and 20 μl assay buffer followed by 100 μl of the opposite extraction buffer were added in sequence. The samples were vortexed and centrifuged (14,000 rpm, 5 min) and clear supernatant was used for the assay. Each well had 40 μl sample or standard and 80 μl Working reagent. Absorbance was measured at zero and 15 min interval at 565 nm at room temperature. Ratio was calculated using the manufacturers’ equation.