Culturing and Differentiating C2C12 Cells
From Bridges Lab Protocols
Growth 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.
- Split cells normally 1/10 or 1/20 into 10% FBS in DMEM with 1x PSG (see Splitting Cells )
- Try to split at 70-80% confluence. Healthy growing cells will reach confluence every other day after a 2X dilution. Split them as fibroblasts into the final format (12 well plate, 6 well plate etc.)
- Cells normally need to be split every other day and we maintain them in 100 mm dishes, if they are not ready to split, refeed them on the second day.
Differentiation
- When cells reach 80-90% confluence switch to media (DMEM, 1x PSG with 2% horse serum).
- Replenish with fresh horse serum media media every other day.
- Cells should be fully differentiated into myotubes by day 7.
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=cellBiology for more details.
