Dulbecco modified eagle medium (dmem)

HeLa cells (CCL-2, ATCC) were maintained in T-75 flasks (Falcon) at 37°C and 5% CO2 in DMEM (Gibco) supplemented with 4.5 g/L glucose, 1 mM sodium pyruvate, 1× non-essential amino acids (Gibco), 10% (vol/vol) FBS (Sigma) and 100 U/mL penicillin-streptomycin mix (Gibco). 100,000 cells were plated on glass-bottom dishes coated with 10 μg/mL fibronectin (MatTek Corporation) in complete DMEM media. The next day, cells were incubated for 2 h in serum-free and phenol-red free DMEM media containing 0.5% BSA. After starvation, cells were incubated with 1 μM C2-568 and 10 μg/mL transferrin-Alexa488 or 100 ng/mL EGF-Alexa488 for 30 min in starvation media to allow the labeled molecules to get internalized by the cells. Labeled cells were washed twice with PBS and fixed with 4% PFA for 10 min. Fixed cells were then washed with PBS before imaging.

Mn²⁺ quenching of fura-2 fluorescence (Hopf et al., 1996 (link)) was used to assess cellular Ca²⁺ entry in FDB fibers of sedentary control mice and of mice performing 3 or 6 wk voluntary running. Mouse FDB muscles were isolated and incubated in DMEM (Sigma-Aldrich) with 10% fetal calf serum and 0.3% type I collagenase (Sigma-Aldrich) for 2 h at 37°C. Dissociated single muscle fibers were plated on laminin coated glass bottom dishes (Mattek) in DMEM with 10% fetal calf serum and incubated for at least 1 h at 37°C. Fibers were loaded with 4 ng/µl fura-2 AM (Invitrogen/Molecular Probes) for 30 min and then washed with Tyrode’s for 30 min. Fura-2 was excited at 360 nm (i.e., the isosbestic point where fluorescence is independent of Ca²⁺), and emitted fluorescence was measured at 495 nm. Baseline fluorescence was measured for 50 s, after which 1 mM MnCl₂ was added and the signal was followed for 50 s. Fibers were then stimulated with 50 repeated 70-Hz, 350-ms tetani given at 2-s intervals, and the fluorescent signal was followed for 500 s subsequent to stimulation, after which MnCl₂ was washed out. The slope of fluorescence decay was measured for 50 s before and between 100 and 400 s after stimulation and expressed relative to the fluorescent signal at the start of the respective measurement period.