Culturing and Differentiating C2C12 Cells

From ATCC

• 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 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
• 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.
• Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
• Add appropriate aliquots of 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°C.

Differentiating

• Myotube formation is stimulated when the medium is supplemented with 2% horse serum instead of fetal bovine serum.