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 Split cells normally 1/10 or 1/20 into 10% FBS with 0.25% PSG (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.see [[ Splitting Cells ]]*Add 2.0 Try to 3.0 ml of Trypsinsplit at 70-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes)80% confluence.*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 need to detach may be placed at 37°C 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 every other day, if they are not ready 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.

 ==DifferentiatingDifferentiation==*When cells reach 80-90% confluence switch to media with 2% horse serum.*Replenish with fresh media every other day.