Reactive oxygen species (ROS) assay

The intracellular levels of ROS in C2C12 myocytes were measured using a dichlorofluorescein assay, as previously described. (Wang and Joseph 2005). In brief, differentiated C2C12 myocytes in 96-well plates were incubated with 10 µM 2´,7´-dichlorodihydrofluorescein diacetate (H₂DCFDA; Sigma-Aldrich) in Krebs–Ringer buffer solution for 30 min at 37 C. After washing with PBS, the cells were treated for 15, 30, or 60 min with 2, 6, 10, and 20 mM lactate or H₂O₂ 500 mM in phenol red-free Minimum Essential Media. Then, intracellular intensity ROS was monitored at excitation and emission wavelengths of 480 and 520 nm, respectively, or fluorescence images were acquired. The fluorescent images were captured using a Nikon Eclipse TE2000-U fluorescence inverted microscope (Nikon, Tokyo, Japan). Images were acquired using an Olympus C-5060 digital camera attached to Nikon TE2000U inverted microscope with a 4x  objective. Fluorescence intensity was measured using excitation and emission wavelengths at 495 and 529 nm, respectively. Fluorescence intensity was quantified using ImageJ software.