Difference between revisions of "Culturing and Differentiating C2C12 Cells"

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[[Category: Cell Biology]]
 
[[Category: Cell Biology]]
  
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==From ATCC==
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==Growth and Maintenance==
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*IMPORTANT - DO NOT ALLOW CULTURES TO BECOME CONFLUENT.  
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*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.
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*Cultures must not be allowed to become confluent as this will deplete the myoblastic population in the culture.
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*Split cells normally 1/10 or 1/20 into 10% FBS with PSG (see [[ Splitting Cells ]]
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*Remove and discard culture medium.
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*Try to split at 70-80% confluence.
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*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.
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*Cells need to be split every other day, if they are not ready refeed them on the second day.
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*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).
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*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.
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*Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
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*Add appropriate aliquots of the cell suspension to new culture vessels.
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*Inoculate at a cell concentration between 1.5 X 10 exp5 and 1.0 X 10 exp6 viable cells/75 cm2.
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*Incubate cultures at 37°C.
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==Differentiation==
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==Differentiating==
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*When cells reach 80-90% confluence switch to media with 2% horse serum.
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*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.
 
*Myotube formation is stimulated when the medium is supplemented with 2% horse serum instead of fetal bovine serum.
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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.

Revision as of 18:19, 15 August 2012


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
  • Try to split at 70-80% confluence.
  • Cells need to be split every other day, if they are not ready refeed them on the second day.

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.

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.

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