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1. Remove and discard culture medium.
2. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
   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 to detach may be placed at 37°C to facilitate dispersal.
3. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
4. Add appropriate aliquots of the cell suspension to new Bovine Collagen type l coated flasks. (0.03 mg/mL)
5. Incubate cultures at 37°C. Myotubes form at confluency. Differentiation is improved by reducing the concentration of both sera to 2% each

Subcultivation Ratio: 1:2 to 1:4
Medium Renewal: Every 2 to 3 days

Complete growth medium described above supplemented with 5% (v/v) DMSO.  Cell culture tested DMSO is available as ATCC Catalog No. 4-X.

Christian CN, et al. Synapse formation between two clonal cell lines. Science 196: 995-998, 1977. PubMed: 193191

Hay, R. J., Caputo, J. L., and Macy, M. L., Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.

Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.