Intracellular NAD+ and NADH Quantification and ATP Measurements

Intracellular NAD⁺ and NADH were measured with NAD⁺/NADH Quantification Colorimetric Kit (BioVision, Milpitas, CA, USA) according to manufacturer’s instructions with some modifications. Briefly, approximate 0.8 million cells were collected and directly lysed in 200 µl lysis buffer from the assay kit. The volume of reagents in each step was scaling down by 50% and the results were calculated by the freshly prepared standard curve (NADH standards provided by the assay kit). Final NAD⁺ and NADH concentrations were then normalized to the total cell number in each group.

ATP measurements: hASCs were centrifuged, re-suspended in deionized water, and heated immediately in boiling water for 15 min. The mixture was centrifuged, and ATP-containing supernatant was collected. Upon measurement, 10 µl of ATP solution was mixed with 100 µl of the luciferin-luciferase reagent (Sigma-Aldrich), and the bioluminescent signal was measured using an Orion Microplate Luminometer (Bad Wildbad, Deutschland). To determine the glycolytic ATP ratio, cells were cultured with or without glycolysis inhibitor 2-Deoxy-D-glucose (2-DG, 5 mM) for 48 h and then the ATP was measured. The ratio of glycolytic ATP was calculated by the delta value of total ATP and 2-DG treated ATP normalized to total ATP.