When skeletal muscle is stretched or injured, satellite cells are activated to enter the cell cycle, and this process could be mediated by hepatocyte growth factor (HGF) and nitric oxide (NO) as revealed by primary culture technique. In this system, which was originally developed by Allen et al. [Allen, R. E., Temm-Grove, C. J., Sheehan, S. M., & Rice, G. (1997). Skeletal muscle satellite cell cultures. Methods Cell Biol., 52, 155-176], however, some populations of satellite cells would receive activation signals during the cell isolation procedure; the high baseline level of activation diminishes the magnitude of the observed effect of HGF and NO. In this study, we modified the cell isolation procedure by lowering pH of muscle and isolation media from 7.2 (original) to 6.5. This modification was designed to block the activation signal generation, based on our previous observations that the satellite cell activation in response to mechanical stimulation only occurred between pH 7.1 and 7.5. Satellite cells prepared at low-pH showed a low baseline level of activation in bromodeoxyuridine incorporation and MyoD expression assays on control cultures, and demonstrated a large activation response to mechanical stretch, exogenous HGF and NO donor. Cell yield and myogenic purity were not affected by the modifications. The low-pH procedure could provide an improved satellite cell model for in vitro activation experiments.