Culturing and Differentiating C2C12 Cells: Difference between revisions

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~~==From ATCC==~~

[[Category: Cell Culture]]

*IMPORTANT - DO NOT ALLOW CULTURES TO BECOME CONFLUENT.

[[Category: Tissue Culture]]

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

[[Category: Cell Biology]]

*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.

==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.

==~~Differentiating~~==

==Differentiation==

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

*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.

@@ Line 1: / Line 1: @@
-==From ATCC==
+[[Category: Cell Culture]]
-*IMPORTANT - DO NOT ALLOW CULTURES TO BECOME CONFLUENT.
+[[Category: Tissue Culture]]
-*Cultures must not be allowed to become confluent as this will deplete the myoblastic population in the culture.
+[[Category: Cell Biology]]
-*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.
+==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.
-==Differentiating==
+==Differentiation==
-*Myotube formation is stimulated when the medium is supplemented with 2% horse serum instead of fetal bovine serum.
+*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.