Soft–Agar colony Formation Assay    

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Any anchorage–independent growth of tumor cells is estimated by a soft–agar colony formation assay. This protocol provides a general workflow for establishing a soft-agar colony formation assay.

Materials and Reagents

  1. Agarose-LE (Guidechem, catalog number: 9012-36-6 )
  2. DMEM (Life Technologies, Gibco®, catalog number: 11995 )
  3. Crystal violet (Matheson Coleman & BEL: catalog number: B278 )
  4. Fetal bovine serum (FBS) (Life Technologies, Invitrogen™, catalog number: 20140-079 )
  5. Phosphate buffered saline (PBS) (Life Technologies, Gibco®, catalog number: 14040 )
  6. Agar
  7. Ethanol
  8. Culture media (see Recipes)


  1. 60 mm culture dishes (Thermo Fisher Scientific, catalog number: 353002 )
  2. Water bath
  3. Incubator


  1. Cells that have been knocked down or been treated with other procedures like drugs or some interferences are harvested and pipetted well to become single-cell suspension in complete culture media in a given concentration (such as 1 x 106/ml).
  2. They are then put at room temperature and should be ready to use. At the same time, pre-warm 10% FBS DMEM at 37 °C, ready to use.
  3. Melt 4% agar (in glass bottle) by microwave and keep warm in 56 °C water bath, ready to use (remember don’t melt the gel too many times, you will lose water and concentrate the gel).
  4. Make bottom layer. Make 5 ml of 10% FBS DMEM containing 0.75% agar (the volume of 4% agar was calculated by: 5 ml multiply 0.75%=X multiply 4%). Therefore, you need to quickly take about 0.9 ml 4% agar from the bottle in water bath and mix it with 4.1 ml pre-warmed of 10% FBS DMEM well and put the mixture into 60-mm culture dish in the flow hood. Wait a few minutes for it to become solid.
  5. Make the top layer. It contains 3 x 104 cells in 3 ml of 10% FBS DMEM and 0.36% agar.
    Dilute the single-cell suspension to 3 x 104 (or 1.5 x 104) in 3ml of 10% FBS DMEM (1 x 104/ml). You need to mix 2.73 ml pre-warmed 10% FBS DMEM cell suspension with 270 μl of 4% pre-warmed agar (56 °C). You have to do this fast and no bubble. To avoid this problem, you can make 6 ml (double) of this mixture and put 3 ml on the top of bottom layer. The best way to do this is to use 1 ml pipette tip to add from one side. The mixture will flow through the whole bottom layer. Do not shake the dish. It will become solid within seconds.
  6. Now the dish contains 2 layers. Mark the dish and put it at 37 °C incubator for 3 weeks.
    Then the colonies were stained with 0.04% crystal violet-2% ethanol in PBS. Photographs of the stained colonies should be taken.
    Note: 4% Agar should be prepared in DD water then autoclave it.
  7. If the colony number is too high to count, you can decrease cells to a total of 1.5 x 104. This would be easy to count.
  8. Before you mix agarose gel with medium and cells. You should get pipette, tips ready to use, and perform it as fast as possible.


  1. Culture media
    10% FBS


This protocol was adapted from Kakuguchi et al. (2010).


  1. Kakuguchi, W., Kitamura, T., Kuroshima, T., Ishikawa, M., Kitagawa, Y., Totsuka, Y., Shindoh, M. and Higashino, F. (2010). HuR knockdown changes the oncogenic potential of oral cancer cells. Mol Cancer Res 8(4): 520-528.
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Liu, F. (2012). Soft–Agar colony Formation Assay. Bio-protocol 2(13): e220. DOI: 10.21769/BioProtoc.220.

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what antibiotics should be added in the agarose when making experiments?
9/11/2014 3:10:56 AM Reply
Kumar Saurabh
University of Louisville
You used agar or agarose-LE?
9/2/2014 12:30:33 PM Reply
Shane Wei
Baylor College of Medicine
Hi, this protocol works pretty well for my cells. But I'm wondering how you stain the colonies by crystal violet. Should I keep the dishes in 37 degree incubator with this dye? How long should I stain the colonies? Some other protocols say that the crystal violet should be prepare just in ddwater without ethanol. What's your opinion? And is it better to wash the colonies after staining?
Thanks very much.
3/31/2014 5:17:24 PM Reply
FengZhi Liu
School of Biomedical Sciences, Thomas Jefferson University, USA

Hi, Shane,
The purpose to stain the colonies is to count it easily and accurately, then take pictures for data documentation. Room temperature is fine. But 37C also is fine since the cells function well and can intake the dye fast. Thus, you should check it frequently. Once you find the agar also is stained, that means the time is too long. I stained it at room temperature. The time cannot be too long. I just watched it, once I found the agar became stained, I used absorbent paper to remove extra dye because the soft agar is fragile. I took pictures before and after staining. No staining is better than staining since the agar always is stained.
I do not think that preparing the crystal violet with ddwater is a good idea. The isotonic status to the colonies is better.

4/12/2014 5:30:12 AM

Shane Wei
Baylor College of Medicine

Hello, your protocol doesn't mention the addition of media. After 2-3 weeks incubation, what if the top gel gets dry and shrunk?

4/14/2014 1:41:32 PM

FengZhi Liu
School of Biomedical Sciences, Thomas Jefferson University, USA

I would like to know if you got dried or shrunk gel after 3 weeks. If it is, please check your incubator if there is a tray with autoclaved water to keep your incubator 95% humidity. I have one experience to share with you. My first result was very good. the colonies were formed in the soft agar. One early morning, mo boss came and saw the results. He was excited and happy. At the same time, he assumed that the top of gel would become dry two days later( just be 3 weeks). He added some media. When I came three hour later, I saw the colonies were swimming in the plate: the soft agar status had been damaged.

Remember the top of soft agar would be dry since the incubator is not a moisture environment. If you forgot to add water in your incubator, it will happen. Under this situation, you may need add one or two drops of media to try to save your results. I do not have this experience.

4/19/2014 7:30:43 AM

FengZhi Liu
School of Biomedical Sciences, Thomas Jefferson University, USA

It is agarouse-LE.

9/2/2014 3:36:31 PM

Aygun Azadova
Debrecen University

Hello FengZhi
Could you explain me the 8th step-Before you mix agarose gel with medium and cells. You should get pipette, tips ready to use, and perform it as fast as possible.Why is it the last place I couldnt get this step well.Thanks in advance.

4/12/2016 3:21:34 PM

Lisha Chen
I would like to know whether I need 2x culture media when I mix the media and the cell suspension.
4/6/2013 9:40:03 PM Reply
FengZhi Liu
School of Biomedical Sciences, Thomas Jefferson University, USA

If the cell is in pellet form, you need 1X media. If the cell already is in some volume media, such as 10 mL 10% RPMI, you only need 1X media, too. Doing so only dilutes cell concentration. Unless you need to add two kinds of media at same time to the cell pellet, under that situation, you need 2X(20%) RPMI, and 0X(0%) RPMI to become 10% RPMI. Remember the "X" means serum concentration.

4/9/2013 10:50:20 AM

Markus Oelze
University of Greifswald

I tried this (with some modifications: Other media, using a 12-well plate and so on). I read somewhere to put some Media on top an change it every 3 days. At first it works just fine, colonies are forming. But after approximately 5 days, the colonies just die. Do you have any idea, why this would happen?

Thank you for your help in advance!
3/26/2013 5:16:52 AM Reply
FengZhi Liu
School of Biomedical Sciences, Thomas Jefferson University, USA

Hi I had not done this way to keep colonies. But I guess the ratio of colony size to media may be the cause. This is similar to keep cells in a flask. You either threw away some cells or decrease the serum concentration in the media.
It is the same to bacteria growth curve.

4/9/2013 10:57:52 AM

we can use any culture media (RPMI-1640) instead of DMEM
8/29/2012 4:03:33 AM Reply
FengZhi Liu
School of Biomedical Sciences, Thomas Jefferson University, USA

It depends on the primary media for the interested cells. If your cells only can grow in RPMI-1640,just use it.

9/5/2012 2:36:47 PM