Thank you for your inquiry Paul and we apologize for the delayed response. Following this protocol, myoblast alignment and primary fusion is seen at around 24h after addition of DM (as you described) and we usually see mature myotubes by 48-72 h. The down side with myotubes prepared from primary myoblast is that they are not sustainable for more than 48-72h after complete myotube formation, i.e 96-120h after the initial addition of DM. This is probably due to the continuous contraction that occurs once myotubes have matured which leads to their detachment from the culturing surface. If what you are observing is cell death prior to myotube formation or during the process of differentiation you first want to exclude the possibility of contamination which could be coming from the differentiation media or any of the other culturing components. Make sure that the horse serum is filtered prior to its addition to the DMEM and make sure that your horse serum is not old (use within 6 months of opening even if aliquots are frozen at -20°). If you are still experiencing the same problem assuming that you are following this protocol and using the referenced reagents, and assuming that you are confident that what you are culturing are myoblasts, one thing that comes to mind is the confluency of the cells before addition of DM. Too high confluency can lead to such results. First of all if the cells are too crowded, this will impose a stressful environment which may lead to their death. Add to that the increase in cell size and elongation that occurs during the alignment process during differentiation, so if there is not enough available room for the cells to comfortably elongate they will start pushing against each other which will lead to their detachment and death. Perhaps consider plating your cells at a lower confluence at the time of DM addition.
Regarding the horse serum in the DM, we have previously used it at 10% which also works fine, however we observed a delay in the differentiation kinetics. Also under such conditions we did not have to change the DM as frequently as the case in the 2%, other than that it worked fine.
You are doing right by heat inactivating your horse serum as it is advisable for better differentiation results, we didn’t mention that in the protocol because the horse serum that we use and is referenced in the protocol is already pre-heat inactivated however we probably should have included a note on that matter.
As far as the sodium pyruvate content, both the F-10 and DMEM that are referenced in this protocol and are used in preparation of MGM and DM respectively contain sodium pyruvate.
We hope this is helpful however let us know if you have any other concerns or your current ones aren’t properly addressed.