Original research article

The authors used this protocol in:
Mar 2009
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Culture, Differentiation and Transfection of C2C12 Myoblasts    

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Abstract

C2C12 myoblasts are commonly used in biomedical laboratories as an in vitro system to study muscle development and differentiation. This protocol explains the basic procedures of culture, transfection and differentiation of C2C12 myoblast cells.

Materials and Reagents

  1. C2C12 myoblasts
  2. DMSO (Sigma-Aldrich, catalog number: 472301 )
  3. Fetal bovine serum (FBS)
  4. Horse serum
  5. DMEM (high glucose) (Life Technologies, Invitrogen™, catalog number: 11965142 )
  6. P/S solution
  7. Fugene HD (FHD) (Roche Diagnostics, catalog number: 04709691001 )
  8. Growth media (see Recipes)
  9. Transfection mix (see Recipes)
  10. Freezing media (see Recipes)
  11. Differentiation media (see Recipes)

Equipment

  1. Standard tabletop centrifuges
  2. Water bath
  3. CO2 incubator
  4. 100 mm culture dishes
  5. Eppendorf tube

Procedure

  1. Grow cells from frozen stock
    1. Briefly thaw cells in a 37 °C pre-warmed water bath.
    2. Once cells are thawed, pipette into Eppendorf tube and spin for 5 min at 1,000 rpm. Aspirate media. Resuspend cells in 10 ml growth media and plate in 100 mm dish.
    3. Split the cells when they grow to 80% confluency.
    4. Refreeze the cells: freeze the cells in freezing media.

  2. Passage cells
    1. Once cells reach 80% confluency, split as 1:40 to a new dish. 3-4 days later, it will be 80% confluency again.
    2. Never let cells grow confluency. They will differentiate.

  3. Transfection
    1. Day 0: seed cells (low density, < 50%).
    2. Day 1: transfection: (optimum 20% FBS, NO P/S).
    3. Day 2: change media (growth media for regular growth or differentiation media for differentiation purpose).
    4. 4-5 days for complete differentiation under confluency.
    5. 3-4 days for complete differentiation under starvation media, but growth is restricted.
    Example:
    1. For 6-well plate: Seed 100,000-150,000 cells total in all 6 plates (10-15,000/cm2).
    2. Transfect 1 μg DNA/6-wells (total).

Recipes

  1. Freezing media
    50% FBS
    10% DMSO
    40% Growth media
  2. Growth media for C2C12 cells
    DMEM
    20% FBS
    1% P/S
  3. Transfection mix
    FHD (1 μg DNA: 4 μl FHD)
  4. Differentiation media
    DMEM
    1% horse serum
    1% P/S

Acknowledgments

This protocol was developed in the Michael Granato Lab at University of Pennsylvania, Philadelphia, USA, and this work was supported by NIH grant R01HD037975.

References

  1. Jing, L., Lefebvre, J. L., Gordon, L. R. and Granato, M. (2009). Wnt signals organize synaptic prepattern and axon guidance through the zebrafish unplugged/MuSK receptor. Neuron 61(5): 721-733.  
  2. Lefebvre, J. L., Jing, L., Becaficco, S., Franzini-Armstrong, C. and Granato, M. (2007). Differential requirement for MuSK and dystroglycan in generating patterns of neuromuscular innervation. Proc Natl Acad Sci U S A 104(7): 2483-2488. 
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Jing, L. (2012). Culture, Differentiation and Transfection of C2C12 Myoblasts. Bio-protocol 2(10): e172. DOI: 10.21769/BioProtoc.172.
Q&A
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If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

Chandler Walker
Indiana University
For this particular protocol, we have found the cells grow well and look healthy with 10% FBS in DMEM instead of 20%. Though the cells do grow a bit faster and are somewhat more robust with 20% serum, 10% serum does not considerably change the behavior of the cells, allowing for saving of costs of serum, which are often high. We have also found that if differentiation is the goal of the experiment, we get the best results when we first let the cells grow to confluence and then switch to low serum medium. By allowing them to initiate some differentiation on their own upon confluence, the behavior of the cells toward differentiation and end phenotype are more consistent than when differentiation is initiated at 80% confluence.
11/30/2018 5:21:01 AM Reply
li jin
shanghai university
i'm sorry! Me poor speed
5/28/2014 10:21:43 AM Reply
li jin
shanghai university
hello! I have some questions of the differentiation of C2C12. The first month after i got the cell,i found in easy to differentiate to myotube after i replace the differentiation medium(DMEM+2%HS+1%PS). but about the second,it very hard for me to see the myotube, and after replace the DM, i often see many dead cells.(i havn't use gelatin). It's a trouble for me. can you give me some supports. thank you!
5/28/2014 10:20:43 AM Reply
Ashok Mandala
CSIR-IICB
hello every one,

I started working with c2c12 one and half a year ago, initially i am satisfied with my cells and results but recently i am getting lot of problems with black dots in the culture and improper differentiation and my RNA yield is only .4-.6 ug/ul (initially it was 3-5ug/ul). so i want to know what could be the reason for this i am eagerly waiting to solve this problem i have tried many ways but no use. can any one help to get rid of this problem.
this is my media
http://www.lifetechnologies.com/in/en/home/technical-resources/media-formulation.8.html
11/30/2013 2:37:00 AM Reply
Petey H
UCSF
After differentiating the myoblasts into myotubes, how would you remove the non-differentiated myoblasts in order to study only myotubes?
10/14/2013 2:58:39 PM Reply
Lili Jing
Department of Cell and Molecular Biology, University of Pennsylvania, USA

Hey,

I haven't tried to do this myself. But I have heard the following from other people and you may give it a try.
After the myotubes are well formed (usually after 5 days), Aspirate medium, wash with PBS, add trypsin, check myotube detach under microscope. When only myotubes but not un-differentiated cells detach, quicly collect the trypsined myotubes. It needs only 1 to 2 min for myotubes detach.

10/14/2013 5:05:11 PM Reply


Petey H
UCSF

Hello, thank you. I think that is a good idea, and I will give it a try.

10/16/2013 11:51:23 AM Reply


Becky Diebold
Emory University
Have you tried Xtreme Gene HP to transfect these cells? Roche has replaced Fugene products with XtremeGene products. I was wondering if Xtreme Gene HP works the same as Fugene HD? Roche recommends 70-90% confluency before transfecting with XtremeGene HP, but maybe 50% would be better? If you have experience with this, please reply. Thank you
10/1/2013 9:39:38 AM Reply
Lili Jing
Department of Cell and Molecular Biology, University of Pennsylvania, USA

Hi, I haven't tried Xtreme Gene HP. I would think it will work.
For growing C2C12 cells, it is better to be low confluency. The cells udergo differentiation very easily once they are confluent.

10/5/2013 10:00:51 AM Reply


100 mm culture dishes, 6 well plate....pls write the product no with name of company
1/12/2013 11:03:15 PM Reply
Thanks for the earlier details.

I would like to know if you have any immunostaining protocol for c2c12 cells. I would like tostain for Notch in c2c12 myoblasts.

Thanks
10/6/2012 2:36:09 AM Reply
Lili Jing
Department of Cell and Molecular Biology, University of Pennsylvania, USA

I haven't done immunostaining for C2C12 cells. Sorry.

10/8/2012 10:56:16 PM Reply


why do you maintain cells in gelatin coated dishes for differentiation? Are the plastic wares used for culturing c2c12 cells different from the ones that are used for other cell lines say 293-T? I have been using 293-T cells and I am switching over to c2c12 cells for some assays. I would like to get a detailed idea of culturing and maintainin c2c12 cells. Can you pls help me with the same
10/1/2012 5:48:52 AM Reply
Lili Jing
Department of Cell and Molecular Biology, University of Pennsylvania, USA

Gelatin coated plates promote the cell attachment and spreading of myoblasts. C2C12 grow fine on regular plastic plates, but the easy grip plate is better.

The information of these plates can be found:

http://www.thermoscientific.com/ecomm/servlet/productsdetail_11152___12976170_-1

http://www.bdbiosciences.com/ecat/Searchresults.do?pgNum=1&pgSize=&sort=SortOrderDef&check=mainsearchcheck&key=gelatin+plates&mterms=true

10/3/2012 5:18:12 AM Reply


How many passages can these cells hold on to. i.e suppose I have cells of passage no.1, how long can it go before i thaw a fresh vial?

Thanks
Sowmya L
10/1/2012 5:38:39 AM Reply
Lili Jing
Department of Cell and Molecular Biology, University of Pennsylvania, USA

The cells can be used up to passages 35-40. We haven't seen a big difference between the low passage and the high passage.

10/3/2012 5:17:31 AM Reply


Hi,
What do you consider "differentiation under starvation media". Is ist DMEM with 1%FBS or DEMEM without any serum?
7/12/2012 7:17:55 AM Reply
Lili Jing
Department of Cell and Molecular Biology, University of Pennsylvania, USA

Either DMEM with 1% horse serum or without any serum will work. But myotubes seem to be more readily formed in medium containing 1% serum than in serum-free condition.

7/16/2012 8:00:23 PM Reply


Becky Diebold
Emory University

Have you tried Xtreme Gene HP to transfect these cells? Roche has replaced Fugene products with XtremeGene products. I was wondering if Xtreme Gene HP works the same as Fugene HD? Roche recommends 70-90% confluency before transfecting with XtremeGene HP, but maybe 50% would be better? If you have experience with this, please reply. Thank you.

10/1/2013 9:38:14 AM Reply


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