Citrate synthase activity kit

After C₂C₁₂ myotubes were incubated with DMEM containing BDSW at different hardness (0, 100, 500, 1000, 1500, 2000 ppm), or 1 mM AICAR for 6 h, enzyme activity was measured using citrate synthase activity kit and cytochrome c oxidase activity kit (BioVision, Inc. Milpitas, CA, USA), and the NAD⁺ and NADH content was determined using NAD⁺ /NADH quantification kit (BioVision, Inc. Milpitas, CA, USA) in cell lysates (10 μg), respectively, according to the manufacturer's instructions. Using a microplate reader (Model AD200; Beckman Coulter, Brea, CA, USA), citrate synthase (CS) and cytochrome c oxidase (COX) activity were determined using a kinetic program at OD 412 nm and OD 550 nm, respectively. The amount of total NAD⁺ or NADH in the lysates was determined at OD 450 nm. The ratio of NAD⁺ to NADH was calculated by dividing the total NAD⁺ or NADH concentration by the total protein concentration.

Mitochondria were isolated from cells as described above. Mitochondrial complex I, II, and IV activities were measured as described previously (Spinazzi et al, 2012). Aconitase activity was measured using Aconitase Activity Microplate Assay Kit (Abcam). Citrate synthase activity was measured from the same sample using Citrate Synthase Activity Kit (BioVision) and was used to normalize the mitochondrial complex activity.