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Culture, Differentiation and Transfection of C2C12 Myoblasts
C2C12成肌细胞   

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参见作者原研究论文

本实验方案简略版
Neuron
Mar 2009

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. 

简介

C2C12成肌细胞通常在生物医学实验室中用作体外系统以研究肌肉发育和分化。 该协议解释了C2C12成肌细胞的培养,转染和分化的基本过程。

材料和试剂

  1. C2C12成肌细胞
  2. DMSO(Sigma-Aldrich,目录号:472301)
  3. 胎牛血清(FBS)
  4. 马血清
  5. DMEM(高葡萄糖)(Life Technologies,Invitrogen TM,目录号:11965142)
  6. P/S解决方案
  7. Fugene HD(FHD)(Roche Diagnostics,目录号:04709691001)
  8. 生长培养基(参见食谱)
  9. 转染组合(参见配方)
  10. 冻结介质(参见配方)
  11. 分化介质(参见配方)

设备

  1. 标准台式离心机
  2. 水浴
  3. CO <2>孵化器
  4. 100毫米培养皿
  5. Eppendorf管

程序

  1. 从冷冻库存生长细胞
    1. 在37℃预热水浴中简单解冻细胞
    2. 一旦细胞解冻,吸管移入Eppendorf管中,并以1,000rpm旋转5分钟。 吸出介质。 重悬细胞在10毫升生长培养基和平板在100毫米的菜。
    3. 当细胞增长到80%汇合时分裂细胞。
    4. 重新冻结细胞:在冷冻培养基中冻结细胞

  2. 通道细胞
    1. 一旦细胞达到80%汇合,分割为1:40到一个新的菜。 3-4天后,再次达到80%汇合
    2. 不要让细胞生长汇合。 他们会区分。

  3. 转染
    1. 第0天:种子细胞(低密度,<50%)
    2. 第1天:转染:(最佳20%FBS,无P/S)
    3. 第2天:更换培养基(用于常规生长的培养基或用于分化目的的分化培养基)
    4. 融合后完全分化4-5天
    5. 3-4天在饥饿培养基下完全分化,但生长受限
    例:
    1. 对于6孔板:在所有6个板(10-15,000/cm 2 )中种子100,000-150,000个细胞。
    2. 转染1μgDNA/6孔(总)

食谱

  1. 冻结介质
    50%FBS
    10%DMSO
    40%生长培养基
  2. C2C12细胞的生长培养基
    DMEM
    20%FBS
    1%P/S
  3. 转染混合物
    FHD(1μgDNA:4μlFHD)
  4. 区分媒体
    DMEM
    1%马血清
    1%P/S

致谢

该协议是在美国宾夕法尼亚大学的Michael Granato实验室开发的,这项工作由NIH授权R01HD037975支持。

参考文献

  1. Jing,L.,Lefebvre,J.L.,Gordon,L.R.and Granato,M。(2009)。 Wnt信号通过斑马鱼拔出/MuSK受体组织突触模式和轴突指导。 < em> Neuron 61(5):721-733。  
  2. Lefebvre,J.L.,Jing,L.,Becaficco,S.,Franzini-Armstrong,C.and Granato,M。(2007)。 MuSK和肌管蛋白聚糖在产生神经肌肉神经支配模式方面的差异要求。 Natl Acad Sci USA 104(7):2483-2488。 
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jing, L. (2012). Culture, Differentiation and Transfection of C2C12 Myoblasts. Bio-protocol 2(10): e172. DOI: 10.21769/BioProtoc.172.
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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


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


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


望 远
西北农林科技大学
4/17/2013 6:54:51 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


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


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


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


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