A. Reagent Reconstitution and General Consideration: Reconstitute NAD Cycling Enzyme Mix with 220 µL NAD Cycling Buffer. Reconstitute NADH developer with 1.2 mL of ddH2O. Pipette up and down several times to completely dissolve the pellet into solution (don't vortex). Aliquot enough NAD Cycling Enzyme mix (2 µL per assay) for the number of assays to be performed in each experiment and freeze the stock solution immediately at -70 °C for future use. The enzymes are stable for up to 2 months at -70 °C after reconstitution. Reconstitute NADH standard with 200 µL pure DMSO to generate 1 nM/µL NADH standard solution. Ensure that the NAD Cycling Buffer is at room temperature before use. Keep other enzymes on ice during the assay and protect from light. B. Sample Preparation:
1. For cell samples*, wash cells with cold PBS. Pellet 2X 10 5 cells for each assay in a micro-centrifuge tube (2000 rpm for 5 min). Extract the cells with 400 µL of NADH/NAD Extraction Buffer by freeze/thaw two cycles (20 min on dry-ice, then 10 min at room temperature), or homogenization. Vertex the extraction for 10 sec. Spin the sample at 14000 rpm for 5 min. Transfer the extracted NADH/NAD supernatant into a labeled tube.
2. For tissue samples*, weight approx. 20 mg tissue, wash with cold PBS, homogenize with 400 µL of NADH/NAD Extraction Buffer in a micro-centrifuge tube. Spin the sample at 14000 rpm for 5 min. Transfer the extracted NADH/NAD supernatant into a new tube. Note: Cell or tissue lysates may contain enzymes that consume NADH rapidly. We suggest to remove these enzymes by filtering the samples through 10 Kd molecular weight cut off filters before performing the assay. C. NADH/NAD
1. Standard Curve: Dilute 10 µL of the 1 nM/µL NADH standard with 990 µL NADH/NAD Extraction Buffer to generate 10 pM/µL standard NADH (Note: diluted NADH solution is unstable, must be used within 4 hours). Add 0, 2, 4, 6, 8, 10 µL of the diluted NADH standard into labeled 96-well plate in duplicate to generate 0, 20, 40, 60, 80, 100 pM/well standard. Make the final volume to 50 µL with NADH/NAD extraction buffer. Samples: To detect total NADt (NADH and NAD), transfer 50 µL of extracted samples into labeled 96-well plate in duplicates. (Note: We recommend performing several different sample dilutions with the Extraction Buffer to ensure the readings fall in the standard curve range). To detect NADH, NAD needs to be decomposed before the reaction. To decompose NAD, aliquot 200 µL the extracted samples into eppendorf tubes. Heat to 60 °C for 30 min in a water bath or a heating block. Under the condition, all NAD will be decomposed, while NADH will still be intact. Cool samples on ice. Quick spin the samples to remove precipitates if precipitation occurs.