Culturing and Differentiating C2C12 Cells: Difference between revisions

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==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 with PSG (see [[ Splitting Cells ]]

*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.

*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 need to be split every other day, if they are not ready refeed them on the second day.

*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 with 2% horse serum.

*When cells reach 80-90% confluence switch to media (DMEM, 1x PSG with 2% horse serum).

*Replenish with fresh media every other day.

*Replenish with fresh horse serum media media every other day.

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

*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.

@@ Line 5: / Line 5: @@
 ==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 with PSG (see [[ Splitting Cells ]]
+*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.
+*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 need to be split every other day, if they are not ready refeed them on the second day.
+*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 with 2% horse serum.
+*When cells reach 80-90% confluence switch to media (DMEM, 1x PSG with 2% horse serum).
-*Replenish with fresh media every other day.
+*Replenish with fresh horse serum media media every other day.
-*Myotube formation is stimulated when the medium is supplemented with 2% horse serum instead of fetal bovine serum.
+*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.