Original research article

The authors used this protocol in:
Jan 2013

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Soft Agar Anchorage-independent Assay    

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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


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


  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.


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


  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.
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: 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
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