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Soft Agar Anchorage-independent Assay
软琼脂锚着非依赖性分析   

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Abstract

Chronic inflammation drives initiation of hepatocellular carcinoma (HCC), but the underlying mechanisms linking inflammation and tumor formation remain obscure. In this study, soft agar anchorage-independent assay were used to determine tumor transform activity of hepatoma cells with ISX over expression or knockdown in vitro.

Materials and Reagents

  1. HCC cells (Hep G2: ATCC® HB-8065TM and Hep 3B: ATCC® HB-8064TM)
  2. ISX fusion GFP expression plasmid or ISX shRNAi
  3. Agarose-LE (MDBio)
  4. 2x MEM no phenol red (Gibco)
  5. 100x NEAA (Gibco)
  6. FBS (Gibco)
  7. 10x PBS (MDBio)
  8. Crystal violet (Sigma-Aldrich, catalog number: C3866 )
  9. Methanol
  10. Ethanol
  11. ddH2O

Equipment

  1. 6 well culture dishes (Greiner Bio-One GmbH)
  2. Water bath
  3. Cell counter
  4. 37 °C incubator

Procedure

  1. HCC cells transfected with ISX fusion GFP expression plasmid or ISX shRNAi and then were selected to stable clones.
  2. The stable HCC clones were then grown in MEM culture medium supplemented with 10% FBS and 1x NEAA according to ATCC guidelines.
  3. Prepare 0.6% and 1.2% agar in ddH2O by autoclave and keep warm in 65 °C water bath.
  4. Making bottom agar: adding 1 ml of 2x MEM culture medium with 20% FBS to 1 ml 1.2% agar. After well mixing, the mixture was put into one well of six-well culture dish to form bottom gel layer.
  5. The stable HCC cells were harvested and counting the cell numbers. Dilute the cells to 1 x 104 cells per ml in 2x MEM with 20% FBS. Adding 1 ml diluted HCC stable cells into 1 ml 0.6% agar. After well mixing, the mixture were put on the top of bottom agar per well. The cell-agar mixture became solid phase at 37 °C incubator for 30 minutes. Then, 2 ml 10% FBS MEM culture medium were added on the top agar in each well.
  6. These dishes were then cultured at 37 °C incubator for 2 weeks and changed the culture medium for each 3 days.
  7. Colonies were visualized by staining with 0.05% crystal violet-75% ethanol or 40% Methanol to 0.45 μm filter. Colonies larger than 0.5 mm were counted.


Acknowledgments

This protocol was adapted from Hsu et al. (2013).

References

  1. Hsu, S. H., Wang, L. T., Lee, K. T., Chen, Y. L., Liu, K. Y., Suen, J. L., Chai, C. Y. and Wang, S. N. (2013). Proinflammatory homeobox gene, ISX, regulates tumor growth and survival in hepatocellular carcinoma. Cancer Res 73(2): 508-518.

简介

慢性炎症驱动肝细胞癌(HCC)的引发,但是连接炎症和肿瘤形成的基础机制仍然是模糊的。 在该研究中,使用软琼脂贴壁非依赖性测定来测定具有ISX的肝癌细胞的肿瘤转化活性超过表达或体外敲低。

材料和试剂

  1. 将HCC细胞(Hep G2:ATCC HB-8065 TM和Hep 3B:ATCC HB-8064 TM sup/)
  2. ISX融合GFP表达质粒或ISX shRNAi
  3. 琼脂糖-EL(MDBio)
  4. 2×MEM不含酚红(Gibco)
  5. 100x NEAA(Gibco)
  6. FBS(Gibco)
  7. 10x PBS(MDBio)
  8. 结晶紫(Sigma-Aldrich,目录号:C3866)
  9. 甲醇
  10. 乙醇
  11. ddH sub 2 O

设备

  1. 6孔培养皿(Greiner Bio-One GmbH)
  2. 水浴
  3. 单元格计数器
  4. 37℃孵育器

程序

  1. 用ISX融合GFP表达质粒或ISX shRNAi转染的HCC细胞,然后选择稳定克隆
  2. 然后根据ATCC指南将稳定的HCC克隆在补充有10%FBS和1×NEAA的MEM培养基中生长。
  3. 通过高压灭菌器在ddH 2 O中制备0.6%和1.2%琼脂,并在65℃水浴中保温。
  4. 制备底部琼脂:加入1ml含有20%FBS的2x MEM培养基至1ml 1.2%琼脂。充分混合后,将混合物放入六孔培养皿的一个孔中以形成底部凝胶层
  5. 收获稳定的HCC细胞并计数细胞数。在具有20%FBS的2x MEM中将细胞稀释至1×10 4个细胞/ml。将1ml稀释的HCC稳定细胞加入1ml 0.6%琼脂中。充分混合后,将混合物置于每孔底部琼脂的顶部。细胞 - 琼脂混合物在37℃培养箱中变成固相30分钟。然后,在每个孔的顶部琼脂上加入2ml 10%FBS MEM培养基
  6. 然后将这些培养皿在37℃培养箱中培养2周,并且每3天更换培养基
  7. 通过用0.05%结晶紫-75%乙醇或40%甲醇至0.45μm滤器染色显现菌落。 计数大于0.5mm的菌落。


致谢

该协议改编自Hsu等人(2013)。

参考文献

  1. Hsu,S.H.,Wang,L.T.,Lee,K.T.,Chen,Y.L.Liu,K.Y.,Suen,J.L.,Chai,C.Y.and Wang,S.N。(2013)。 促炎同源盒基因,ISX,调节肝细胞癌中的肿瘤生长和存活。 Cancer Res 73(2):508-518
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, L. and Hsu, S. (2013). Soft Agar Anchorage-independent Assay. Bio-protocol 3(20): e947. DOI: 10.21769/BioProtoc.947.
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Shih-Hsien Hsu
Graduate Institute of Medicine, Kaohsiung Medical University, Taiwan
The cells (Hep 3B) will die to 40~50% when we sub-cultured it at secondary time at the 700ng/ml G418 dosage. The cells will die to 80~90% at the third subculture. The death cells will dependent on the transfection efficiency. If not so, I suggest you had checked you Hep 3B cells and It maybe had changed its cellular characterization.
12/18/2013 4:12:48 AM Reply
xiao xiao
Sun Yat-Sen University

Thank you !

12/19/2013 11:16:57 PM


xiao xiao
Sun Yat-Sen University
I read your paper and it is interesting. But, how to establish stable cell lines expressing desired gene with vectors ? How to select Hep 3B to stable clones? When I treated Hep 3B cells with G418 from 100 to 1100ug/ml and maintained 7 days(change once 3 days),the cells did not die out.Then after two weeks,10% of cells were still alive at 1100ug/ml. Is Hep 3B resistant to G418 ? I noticed your vectors carry neomycin gene and would you mind tell me the detail method of establishing stable cell lines ? Thank you very much
12/17/2013 9:41:54 PM Reply