Changes

==From ATCCGrowth and Maintenance==*IMPORTANT - DO NOT ALLOW CULTURES TO BECOME CONFLUENT. *Cultures must not be allowed to become confluent as this will deplete the myoblastic population in the culture.*Remove and discard culture medium.*Briefly rinse the cell layer with 0.25Split cells normally 1/10 or 1/20 into 10% FBS in DMEM with 1x PSG (w/vsee [[ Splitting Cells ]]) Trypsin- 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.*Add 2.0 Try to 3split at 70-80% confluence.0 ml of Trypsin-EDTA solution to flask and observe Healthy growing cells under an inverted microscope until cell layer is dispersed will reach confluence every other day after a 2X dilution. Split them as fibroblasts into the final format (usually within 5 to 15 minutes)12 well plate, 6 well plate etc.)*Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult normally need to detach may be placed at 37°C split every other day and we maintain them in 100 mm dishes, if they are not ready to facilitate dispersal.*Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.*Add appropriate aliquots of split, refeed them on the cell suspension to new culture vessels.*Inoculate at a cell concentration between 1.5 X 10 exp5 and 1.0 X 10 exp6 viable cells/75 cm2.*Incubate cultures at 37°Csecond day.

see http://www.stanford.edu/group/blau/protocols/c2lineprotocol.html and http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=crl-1772&Template=Differentiating==*Myotube formation is stimulated when the medium is supplemented with 2% horse serum instead of fetal bovine serumcellBiology for more details.