Eclipse ts100 f microscope

Myoblasts were seeded on 35 mm dishes that contained a 1.5 mm thick collagen coated coverslip (MatTek Corporation, Ashland, MA, USA) in normal growth media. Triplicate groups of cells were exposed to vehicle (DMSO, control) or a combination of 5 mM caffeine, 10 mM 3-methyladenine (3-MA), or 30 μM STO609. Samples were washed in PBS and fixed with 4% paraformaldehyde for 20 min. analyzed using a Nikon Eclipse TS100-F microscope and NIS Elements Br software (Nikon Instruments, Melville, NY, USA). Ten images were randomly captured on each coverslip (replicate sample). The diameter of every myotube (5–10) in each image was measured and used to calculate the mean myotube diameter for each sample replicate (n= 3).

Myoblasts were seeded on 35 mm dishes that contained a 1.5 mm thick collagen coated coverslip (MatTek Corporation, Ashland, MA, USA) in normal growth media. Triplicate groups of cells were exposed to vehicle (DMSO, control) or a combination of 5 mM caffeine, 30 μM STO609, 10 μM KN93 or 10 μM KN92. All possible experimental scenarios were tested using these treatment groups. Protein synthesis analysis was conducted using a Molecular Probes Click-iT^® Plus OPP Alexa Fluor^® 488 Protein Synthesis Assay Kit (Thermo Fisher Scientific, Carlsbad, CA, USA) according to manufacturer’s protocol. Samples were analyzed using a Nikon Eclipse TS100-F microscope and NIS Elements Br software (Nikon Instruments, Melville, NY, USA). Ten images were randomly captured on each coverslip (replicate sample), which were subsequently used to calculate the mean FITC intensity of individual myotubes by creating a region of interest around myotubes in each image. The mean FITC intensity for each image calculated and used to calculate the average intensity for each experimental treatment (n = 3).

Myoblasts were seeded on 35 mm dishes that contained a 1.5 mm thick collagen coated cover-slips (MatTek Corporation, Ashland, MA, USA) in normal growth media. Triplicate groups of myoblasts or myotubes either received vehicle or 5 mM caffeine for 6 h. A terminal transferase dUTP nick end-labeling (TUNEL) assay was conducted to evaluate DNA fragmentation according to the manufacturer’s protocol (Roche Molecular Biochemicals, Pleas-anton, CA, USA). Nuclei were stained with 2 μl/ml of Hoechst 3334 for 30 min at 37 °C. All samples were washed in TBS and fixed with 4% paraformaldehyde for 20 min. Samples were analyzed using a Nikon Eclipse TS100-F microscope with a 40× fluorite objective and NIS Elements Br software (Nikon Instruments, Melville, NY, USA). Cell death was calculated as the percentage of TUNEL positive nuclei compared to total number of Hoechst stained nuclei.