Roche FuGENE® Transfection Reagents）

The C2C12 cell line differentiates rapidly, forming contractile myotubes and producing characteristic muscle proteins. [22953]

Treatment with bone morphogenic protein 2 （BMP-2） cause a shift in the differentiation pathway from myoblastic to osteoblastic. [23427]

Tested and found negative for ectromelia virus （mousepox）.

Cultures must not be allowed to become confluent as this will deplete the myoblastic population in the culture.

Myotube formation is enhanced when the medium is supplemented with 10% horse serum instead of fetal bovine serum.

1. Remove and discard culture medium.

2. 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.

3. 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.

4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.

5. 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.

6. Incubate cultures at 37°C.