Nad nadh assay kit

Intracellular NAD⁺ and NADH levels were measured using the NAD/NADH Assay Kit (ab65348; Abcam, Cambridge, UK) according to the manufacturer’s protocol. Briefly, cells were extracted with the NAD/NADH extraction buffer and filtered through a 10 kD spin column to remove enzymes that consume NADH. To detect the NADH only, decomposition was performed by heating the samples at 60°C for 30 min. As such, NAD⁺ was decomposed while the NADH was intact. The decomposition step was omitted in the detection of total NAD⁺ and NADH. NAD⁺ in the samples was then converted to NADH by adding NADH developer. Concentrations of NADH in the samples were derived from the standard curve. NAD⁺/NADH ratio was calculated as ((total NAD⁺ and NADH) - NADH)/NADH.

Intracellular NAD⁺ and NADH were determined as per the manufacturer's protocol (NAD⁺/NADH Assay Kit, Cat No: ab65348, Abcam) and as described earlier (Manna et al., 2018 (link)). Briefly 2 × 10⁶ control or heat-shocked cells were lysed in NAD⁺/NADH extraction buffer by sonication (five pulses at 15 amp for 15 s). The lysate was centrifuged at 14,000 rpm and the supernatant containing NAD⁺/NADH was filtered through a 10 kDa spin column to get rid of enzymes, which may consume NADH rapidly. To detect the NADH in the sample, a decomposition step was performed by heating the samples at 60°C for 30 min; under this condition, all the NAD⁺ will be decomposed while NADH will be still intact. 100 μl reaction mix was prepared for each standard and samples were plated in duplicates in a clear bottom 96 well plate (Corning, Catalog # CLS3603). The plate was incubated at room temperature for 5 min to convert NAD⁺ to NADH followed by addition of 10 μl NADH developer into each well and incubated at room temperature for 2 h. OD was measured at 450 nm using a plate reader (BioTek Cytation3).

The measurement of NAD⁺ levels was performed using the NAD/NADH Assay Kit (Abcam) as per the manufacturer's instructions. The ovarian tissue was homogenized in lysis buffer and the supernatant was collected after centrifugation. The samples and the NAD⁺ Extraction buffer were pre‐heated at 37°C for 10 min. Then, the NADH Extraction and NADH Reaction Mixture were added to the tissue supernatant and incubated at room temperature for 2 h. Subsequently, the fluorescence intensity was determined in a microplate reader with excitation and emission wavelengths set at 540/590 nm.

NAD/NADH ratio was measured with NAD/NADH assay kit purchased from Abcam. Briefly, HUH6 cells were cultured with or without 5 μM CQ for 96 h. Cells were lysed and processed following manufacturer's instructions as described (49 (link)). The reaction was allowed to develop for 1.5 h, and then absorbance was measured at 450 nm with Multiskan FC microplate reader (Thermo Fisher Scientific, Vantaa, Finland). NAD/NADH ratio was calculated using normalized concentrations by equation ([NADtotal—NADH])/[NADH].