Nad nadh assay kit with wst 8

The ratio of NAD⁺/NADH was measured using the NAD⁺/NADH Assay Kit with WST-8 (Beyotime). To detect the changes of NAD⁺/NADH, ovaries were collected on PD14 and PD15, respectively. To explore the effect of rapamycin intake on NAD⁺/NADH, ovaries were collected on PD15. Thereafter, the collected ovaries were homogenated with the precooled lysis buffer (200 μl). To measure the total amount of “NAD⁺ + NADH,” the alcohol dehydrogenase working solution (90 μl) and chromogenic solution (10 μl) were successively added to the ovarian lysates. To detect the amount of NADH, the ovarian lysates were heated to 60 °C for 10 min, followed by centrifugation. The supernatant (20 μl) was then incubated with 90 μl alcohol dehydrogenase working solution at 37 °C for 10 min. Finally, the reaction system was added with 10 μl chromogenic solution and incubated in dark at 37 °C for 30 min. The absorbance at 450 nm was measured by a microplate reader. The standard curves were produced simultaneously to calculate the contents of “NAD⁺ + NADH” and NADH content. NAD⁺/NADH was calculated according to the formula: (“NAD⁺ + NADH” − NADH)/NADH.

The content of NAD⁺ and ATP of SN4741 cells was measured using a NAD⁺/NADH Assay Kit with WST‐8 (Beyotime, S0175) and Enhanced ATP Assay Kit (Beyotime, S0027), and was measured by spectrometer and luminometer, respectively. Carry out related operations according to the manufacturer's instructions.

The cellular content of NAD⁺ and NADH was determined using a NAD+/NADH Assay Kit with WST-8 (S0175, Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions. In brief, approximately 1 × 10⁶ VMSCs were plated into each well of six-well plates and incubated with 200 μLof NAD+/NADH extraction solution. The NAD+/NADH ratio was calculated based on the NAD+ and NADH standard curves.

NAD_total levels and NAD⁺/NADH were determined using NAD⁺/NADH assay kit with WST-8 (Beyotime, S0175) according to the manufacturer’s instructions. In brief, adherent neurons or cortical tissues were lysed with 400 µl of lysis buffer, and centrifuged at 12,000 ×g for 10 min. 90 µl of alcohol dehydrogenase was added to a 96-well plate. NAD_total levels were obtained by adding 20 µl of the supernatant or standard substance. And NADH levels were obtained by adding 20 µl of the suspension or standard substance after incubating at 60 °C for 30 min and was added to a 96-well plate. Subsequently, 10 µl of chromogenic solution was added to the plate and the mixture was incubated at 37 °C for 30 min. The absorbance values were measured at 450 nm and analyzed on a multimode microplate reader (Tecan Spark). Standard curve was generated and the protein concentration of each sample was measured by BCA protein assay. The amount of NAD⁺ was derived by subtracting NADH from NAD_total.

20 ml of wild-type and mutants Syn2973 grown to an OD₇₃₀ of 1.0 were harvested by centrifugation (8,000 rpm, 3 min, 4 °C) and washed with cold PBS buffer twice. The cells were resuspended in 600 μl NAD⁺/NADH extraction buffer or in 600 μl NADP⁺/NADPH extraction buffer, respectively. Cells were disrupted by an ultrasonic cell disruptor for 10 min at 0 °C. The supernatant was used for NADH, NAD⁺ or NADPH, NADP⁺ determination.
The intracellular NADH, NAD⁺ and NADPH, NADP⁺ were determined by using NAD⁺/NADH Assay Kit with WST-8 (Beyotime Biotech, Shanghai, China) and NADP⁺/NADPH Assay Kit with WST-8 (Beyotime Biotech, Shanghai, China) according to the manufacturer’s instruction, respectively. The concentrations of NADH, NAD⁺ and NADPH, NADP⁺ were calculated according to the NADH and NADPH standard curve, respectively.
The intracellular ATP and ADP concentrations were determined by using the ADP/ATP Ratio Assay Kit (Sigma-Aldrich, USA)[57 (link)] according to the manufacturer’s instruction. The ATP and ADP concentrations were calculated according to the ATP standard curve.

The intracellular rates of NADP⁺ and NADPH were measured using an NAD⁺/NADH Assay Kit with WST-8 (S0175; Beyotime) according to the manufacturer's instructions. In brief, 1 × 10⁶ indicated that PDAC cells were collected and lysed with 200 μL of NADP⁺/NADPH extraction solution. Lysate samples were centrifuged at 12,000 g for 8 min at 4°C to collect the supernatant. The supernatant was then separated into two portions. One portion was heated to 60°C to deplete NADP⁺. Together with G6PDH working solution, the samples were added to 96-well plates and incubated at 37°C for 10 min under dark conditions. The absorbance was then measured at 450 nm.

Transfected U87 and U251 cells were collected to determine the NAD+ levels using an NAD+/NADH assay kit with WST-8 (Beyotime, Shanghai, China) and a Mitochondria/Cytosol Fractionation Kit (BioVision, K256-100 CA, USA) according to the manufacturer’s instructions. In brief, cells (1×10⁶/sample) were lysed and isolated with a Mitochondria/Cytosol Fractionation Kit. To measure the total NAD+/NADH ([NAD_total]), 20 μL of cyto-lysates was added to a 96-well plate. To measure NADH ([NADH]), the lysed cells were incubated at 60 °C for 30 min, and 20 μL was added to a 96-well plate. Subsequently, 90 μL of alcohol dehydrogenase was added and incubated at 37 °C for 10 min. Finally, 10 μL of chromogenic solution was added to the plate, and the mixture was incubated at 37 °C for 10 min. A standard curve was generated and measured at the same time as the samples. The absorbance values were measured at 450 nm and analyzed on a plate reader. The amount of NAD+ was derived by subtracting NADH from the total NAD+/NADH ([NAD+] = [NAD_total] - [NADH]; [NAD+]/[NADH] = ([NAD_total] - [NADH])/[NADH]).