i have been trying to differentiate primary human myoblasts into myotubes. i have early pssage (p2) and old pssage (p14). Hoth with same outocmes.

the myoblasts have been growing in 20% fcs, 0.5% chicken embryo in f10 (hams media). the problem is that when i resuspend them in 5% horse serum low glucose dmem, and plate them in poly lysine and matrigel coated 24 well plates, they do not attach and hence they do not differentiate. they just sit on the bottom in a round shape. Very Few attach but most do not. i seeded 20k.

alternatively, i seeded them in grow media, once aconfouent i add the differentiation media (horse serum dmem) and they detach taking a round shape. have tried many times and it does not work. in my institute they seem to work under former conditions.

will highly appreciate your feedback and help.