Nad nadh ratio assay kit

L6 myotubes were differentiated in in 6-well plates and treated with 60 µM compounds for 24 h in duplicate wells. ADP/ATP Ratio Assay kit (Abcam) and NAD⁺/NADH Ratio Assay kit (Abcam) were used according to the manufacturer’s instructions.

The NAD⁺/NADH ratio was detected in cultured cells with an NAD⁺/NADH ratio assay kit (Abcam, Cambridge, MA, USA) [15 (link)]. Briefly, cells were washed with phosphate-buffered saline (PBS) buffer and lysed with lysis buffer for 15 minutes at room temperature. Then, the NAD and NADH extraction reagents were added and the reaction was proceeded at room temperature for 15 minutes. PBS was used as blank control, and the fluorescence signal was measured after 30 minutes in conditions of excitation (540 nm) and emission (590 nm) with a Varioskan™ Flash Multimode Reader (Thermo Scientific, Waltham, MA, USA). Mitochondrial ROS were measured according to a previously published protocol [15 (link)]. Briefly, cells were washed with Hank's buffered salt solution (HBSS) and incubated with HBSS containing 5 μM Mito SOX (Thermo Scientific, Waltham, MA, USA) at 37°C for 20 minutes. Cells were washed three times with HBSS, and fluorescence signal was detected using a Varioskan Flash Multimode Reader (Thermo Scientific, Waltham, MA, USA) under the conditions of excitation (488 nm) and emission (530 nm).