Jenner-Giemsa staining and measurement of myotube length and diameter

Myotube length and diameter were measured using images obtained through Jenner-Giemsa staining of C2C12 differentiated cells (Veliça and Bunce, 2011). The cultured cells were washed with PBS, fixed with 100% methanol for 5 min, and air dried for 10 min. The Jenner staining solution (Sigma-Aldrich, St. Louis, MO) was diluted 3-fold with 1 mM sodium PBS (pH 5.6); 1 mL of this solution was incubated in the wells for 5 min and then the cells were washed with distilled water. The cells were then incubated for 10 min at 25°C with 1 mL Giemsa stain diluted 1:20 with 1 mM sodium PBS (pH 5.6) and then washed again with water. Cells in each well were observed using a MM-400 microscope (Nikon, Tokyo, Japan) and photographed in nine randomly selected areas using a digital microscope camera JENOPTIK ProgRes speed Xt^core 3 and the ProgRes Image Capture Software (Jenoptik Optical Systems GmbH, Jena, Germany). Myotube length and diameter were measured and quantified using the Image-J software (Scion, Frederick, MD, USA).