Nad nadh colorimetric assay kit

To measure the NAD⁺/NADH ratio in mitochondria, cells were cultured in standard conditions prior to mitochondria isolation that was performed as described above. The NAD⁺/NADH ratio was measured using the NAD⁺/NADH colorimetric assay kit (Abcam, Waltham, MA, USA), according to the manufacturer’s instructions. Briefly, isolated mitochondria were lysed by two freeze/thaw cycles in NAD⁺/NADH Extraction Buffer and vortexed for 10 seconds. After centrifugation, half supernatant was used for measurement of NADt (total amount of NAD⁺ and NADH), and the other half was used for measurement of NADH (after decomposition of NAD⁺ at 60 °C for 30 min). Samples were incubated for 5 min with NAD Cycling Mix, followed by NADH Developer solution for 4 h. Absorbance at 450 nm was then measured. The NAD⁺/NADH ratio was calculated as follows: (NADt - NADH)/NADH.

Cells were seeded in tissue-culture treated 6-well plates and allowed to grow until they reached 80% confluency. Cellular NAD⁺/NADH levels were determined with the NAD/NADH Colorimetric Assay Kit (ab65348, Abcam), according to the manufacturer’s instructions. The assay was performed in 96-well clear flat bottom plates and absorbance at 450 nm was measured in a SpectraMax M2/M2e microplate reader (Molecular Devices) after 2 h incubation at RT with NAD cycling enzyme mix. Total NAD and NADH concentrations were calculated by interpolation to a NADH standard curve. The levels of NAD⁺ were calculated by subtracting NADH from total NAD, and the NAD⁺/NADH were represented.

Liver tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and stained with H&E. Liver lipids were extracted using chloroform/methanol (2:1) as a solvent. Total triglyceride contents of the liver were determined by an enzymatic method (Stanbio Laboratory) (14 (link), 85 (link)). NAD⁺ and NADH were measured using the Abcam NAD/NADH Colorimetric Assay Kit (catalog ab65348) (14 (link)).

NAD⁺/NADH levels in cellular pellets were assessed by the NAD⁺/NADH Colorimetric Assay Kit (Abcam, Hercules, CA, United States, ab65348) protocol. Colour intensity was measured using a POLARstar Omega plate reader (BMG Labtech, Offenburg, Germany).

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)].

Intracellular nicotinamide adenine dinucleotide (NAD⁺/NADH) levels were assessed using the NAD⁺/NADH Colorimetric Assay Kit (Abcam, Hercules, CA, USA, ab65348). Absorbance was measured using a POLARstar Omega plate reader (BMG Labtech, Offenburg, Germany). Each assay was performed in three biological replicates.

BV2 microglia were seeded in a 6-well plate and treated with thymoquinone (2.5–10 µM) for 24 h. Cell lysates were collected with 400 μL of NADH/NAD extraction buffer (Abcam). Quantification of NADH and NAD, and their ratio was carried out with a colorimetric NAD/NADH assay kit (Abcam), according to the manufacturer’s instructions.