Hello!

I am currently conducting experiments on the isolation, culture, and staining of single myofibers from the extensor digitorum longus (EDL) of mice to observe the self-renewal, proliferation, and differentiation of satellite cells.

I am having difficulty interpreting the data. According to references, after 72 hours of culture, the ratio of three states within the cluster is often analyzed: PAX7+/MYOD-, PAX7+/MYOD+, and PAX7-/MYOD+.

However, in my 72-hour culture experiment, I am unable to observe the expected ratios. Instead, all the cells within the cluster show a PAX7+/MYOD+ ratio.

Thinking that the culture duration might be too short, I extended the culture to 5 days, and only then was I able to observe the expected ratios. However, maintaining the fibers in culture for 5 days is very challenging.

Therefore, I would appreciate any advice on how to achieve clear analysis with only 3 days of culture, as suggested by other references.

I will attach the experimental conditions and result images below.

[Mice: ICR, Female, 2~3month]

[myofiber culture media: DMEM high glucose with 20% FBS, 1% P/S, and 10% CEE]

Thank you in advance for any suggestions or insights. I look forward to your feedback!