Culturing and Differentiating C2C12 Cells - Revision history

Davebrid: updated with more media details

2016-12-09T15:34:46Z

updated with more media details

← Older revision		Revision as of 15:34, 9 December 2016
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	==Growth and Maintenance==		==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.		*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==		==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.		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.

Davebrid: added differentiation protocol

2012-08-15T18:19:35Z

added differentiation protocol

← Older revision		Revision as of 18:19, 15 August 2012
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	[[Category: Cell Biology]]		[[Category: Cell Biology]]

	==~~From ATCC~~==		==Growth and Maintenance==
	*IMPORTANT - DO NOT ALLOW CULTURES TO BECOME CONFLUENT.		*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.
	*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 ]]
	*~~Remove and discard culture medium.~~		*Try to split at 70-80% confluence.
	*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.~~		*Cells need to be split every other day, if they are not ready refeed them on the second day.
	*~~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.

			==Differentiation==
			*When cells reach 80-90% confluence switch to media with 2% horse serum.
			*Replenish with fresh media every other day.
			*Myotube formation is stimulated when the medium is supplemented with 2% horse serum instead of fetal bovine serum.

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

Davebrid: added categories

2012-08-08T14:11:24Z

added categories

← Older revision		Revision as of 14:11, 8 August 2012
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			[[Category: Cell Culture]]
			[[Category: Tissue Culture]]
			[[Category: Cell Biology]]

	==From ATCC==		==From ATCC==
	*IMPORTANT - DO NOT ALLOW CULTURES TO BECOME CONFLUENT.		*IMPORTANT - DO NOT ALLOW CULTURES TO BECOME CONFLUENT.

Davebrid: wrote initial protocol

2012-08-08T14:09:57Z

wrote initial protocol

New page

==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.