NAD⁺/NADH Assay Kit (WST-8)

Reagent Preparation

1. NADH standard stock: add 665 μL NADH Solution to 0.5 mg NADH and dissolve completely to obtain a 1 mM NADH stock solution. Aliquot and store at -80°C protected from light; avoid repeated freeze-thaw cycles.

2. NADH standard curve: dilute the NADH stock with NAD⁺/NADH Extract Solution to 0, 0.25, 0.5, 1, 2, 4, 6, 8, and 10 μM. Add 20 μL per well, corresponding to 0, 5, 10, 20, 40, 80, 120, 160, and 200 pmol NADH. Use the 0 μM standard as the blank.

3. ADH working solution: 90 μL is required per standard or sample well. Dilute ADH 1:20 with Reaction Buffer and prepare immediately before use.

Sample Preparation

1. Adherent cells: collect approximately 1 × 10⁶ cells, remove the culture medium, and add 200 μL pre-cooled NAD⁺/NADH Extract Solution.

2. Suspension cells: collect approximately 1 × 10⁶ cells, centrifuge at 600 × g for 5 min, discard the supernatant, and add 200 μL pre-cooled NAD⁺/NADH Extract Solution.

3. Lyse cell samples thoroughly for approximately 10 min at room temperature or on ice. Centrifuge at 12,000 × g for 5-10 min at 4°C and collect the supernatant.

4. Tissue: wash with pre-cooled PBS, weigh 10-30 mg and mince. Add 400 μL NAD⁺/NADH Extract Solution, homogenize thoroughly at room temperature or on ice, centrifuge at 12,000 × g for 5-10 min at 4°C, and collect the supernatant.

Total NAD⁺/NADH Assay

1. Add 20 μL sample supernatant to each sample well; 3-5 replicate wells are recommended. Add 20 μL of the appropriate NADH standard to each standard well.

2. Add 90 μL ADH working solution to each well, mix gently, and incubate at 37°C for 10 min to convert NAD⁺ in the sample to NADH.

3. Add 10 μL Chromogen Solution to each well, mix gently, and incubate at 37°C for 30 min protected from light.

4. Measure absorbance at 450 nm using a microplate reader.

NADH Assay

1. Transfer 50-100 μL sample supernatant to a centrifuge tube and heat at 60°C for 30 min to decompose NAD⁺. Cool to room temperature and briefly centrifuge.

2. Use 20 μL of the heated sample and perform Steps 2-4 of the Total NAD⁺/NADH Assay.

Calculation

Determine total NAD⁺/NADH and NADH amounts from the NADH standard curve. NAD⁺ = total NAD⁺/NADH - NADH; NAD⁺/NADH ratio = NAD⁺ amount ÷ NADH amount. Multiply by the dilution factor and normalize to cell number, tissue weight, or protein content as required.