Cytation hybrid

Cells were seeded into 96-well plates at a density of 2500 cells per plate. CCK-8 reagent was added to each well according to the manufacturer’s instructions (Dojindo, MD, USA). After incubation for 2–4 h at 37 °C, the absorbance at 450 nm was measured for each sample well using a microplate reader (BioTek Cytation Hybrid, Agilent, CA, USA).

The levels of NAD⁺ and NADH were determined using a WST-8-based NAD⁺/NADH assay kit (Beyotime, S0175, Beijing, China). In brief, after mechanical stretching, 1 × 10⁶ SCs were washed twice with cold PBS and further lysed using NADH/NAD extraction buffer. The samples were centrifuged at 12,000 g for 10 min to collect the supernatant. For the measurement of total NAD⁺/NADH, 20 μL of cell lysis supernatant was added with 90 μL of working solution for incubation in the dark at 37 °C for 10 min. Then, 10 μL of color-developing agent was added to the plate for further incubation at 37 °C for 30 min. The absorbance was measured at 450 nm using a microplate reader (BioTek Cytation Hybrid, Agilent, CA, USA). For the measurement of NADH, the supernatant was incubated at 60 °C for 30 min first and then processed into the procedure as described above. The levels of NAD⁺ were derived by subtracting NADH from total NAD⁺/NADH.