Clonogenic Assay   

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Original research article

A brief version of this protocol appeared in:
Nature Protocols
Dec 2006


Clonogenic assays serve as a useful tool to test whether a given cancer therapy can reduce the clonogenic survival of tumor cells. A colony is defined as a cluster of at least 50 cells that can often only be determined microscopically. A clonogenic assay is the method of choice to determine cell reproductive death after treatment with ionizing radiation, but can also be used to determine the effectiveness of other cytotoxic agents. The following protocol has been modified from a published version (Franken et al., 2006).

Materials and Reagents

  1. Cell culture medium
  2. Phosphate buffered saline (PBS)
  3. Fetal bovine serum (FBS)
  4. Trypsin/ EDTA (Life Technologies, Invitrogen™, catalog number: 25200-056 )
  5. Crystal violet (Sigma-Aldrich, catalog number: C3886 )
  6. Methanol (Sigma-Aldrich, catalog number: 34860 )
  7. Glacial acetic acid (Sigma-Aldrich, catalog number: 320099 )
  8. Fixation solution
  9. Colony fixation solution (see Recipes)
  10. Crystal violet solution (see Recipes)


  1. Cell culture petri dishes or six-well plates (Thermo Fisher Scientific, catalog number: 08-772-1B )
  2. Hemocytometer (Hausser Bright-Line) (Thermo Fisher Scientific, catalog number: 02-671-10 )
  3. Stereomicroscope (e.g., Nikon Eclipse, model: TS100 )
  4. Hemocytometer
  5. Incubator


  1. Cell preparation:
    1. Culture the cells according to the requirement (e.g., GBM cell lines, U87, U251, SF188, etc).
    2. Remove medium, and then rinse cells with 10 ml PBS.
    3. Add 4 ml 0.25% trypsin to the cells and incubate at 37 °C for 1-5 min until the cells appear round.
    4. Add 10 ml medium with 10% FBS, and detach the cells by pipetting.
    5. Count the cells using a hemocytometer.
      Note: It is critical to get a relatively accurate number for the cells.
    6. Prepare desired seeding concentration, and then seed cell into dishes or 6-well plates.

  2. Assay setup:
    Cell can be plated either before or after the treatment.
    1. Plating before treatment:
      1. Harvest cells and plate an appropriate number of cells per dish or per well on a 6-well plate, at least in duplicate. The number of cells for seeding should be determined by the aggressiveness of the treatment. Incubate cells for a few hours in a CO2 incubator at 37 °C and allow them to attach to the plate/dish.
      2. Treat the cells as necessary with chemicals (e.g., 1-100 μM), radiation (e.g., 2-10 Gy) or a combination of both.
      3. Incubate the cells in a CO2 incubator at 37 °C for 1-3 weeks until cells in control plates have formed colonies that are of a substantially good size (50 cells per colony is the minimum for scoring).
    2. Plating after treatment:
      1. Harvest cells after treatment. Fifty or up to 50 x 104 cells can be plated. Prepare serial dilutions with different numbers of cells, should the effects of the treatments be unclear. For radiation treatment, the cells can be plated immediately after treatment or re-plated later. It is always better to keep the cells on ice before re-plating.
      2. Incubate the cells in a CO2 incubator at 37 °C for 1-3 weeks until cells in control plates have formed colonies with substantially good size (50 cells per colony is the minimum for scoring).

  3. Fixation and staining:
    1. Remove medium, and then rinse cells with 10 ml PBS.
    2. Remove PBS and add 2-3 ml of fixation solution and leave the dishes/plates at room temperature (RT) for 5 min.
    3. Remove fixation solution.
    4. Add 0.5% crystal violet solution and incubate at RT for 2 h.
    5. Add 10 ml medium with 10% FBS, and detach the cells by pipetting.
    6. Remove crystal violet carefully and immerse the dishes/plates in tap water to rinse off crystal violet.
    7. Air-dry the dishes/plates on a table cloth at RT for up to a few days.

  4. Data analysis:
    1. Count number of colonies with a stereomicroscope.  
    2. Calculate plating efficiency (PE) and surviving fraction (SF).
      PE = no. of colonies formed/ no. of cells seeded x 100%
      SF = no. of colonies formed after treatment/ no. of cells seeded x PE


  1. Colony fixation solution
    Acetic acid/methanol 1:7 (vol/vol)
  2. Crystal violet 0.5% solution


This protocol has been modified from a published version (Franken et al., 2006).


  1. Franken, N. A., Rodermond, H. M., Stap, J., Haveman, J. and van Bree, C. (2006). Clonogenic assay of cells in vitro. Nat Protoc 1(5): 2315-2319.
  2. Mueller, S., Yang, X., Sottero, T. L., Gragg, A., Prasad, G., Polley, M. Y., Weiss, W. A., Matthay, K. K., Davidoff, A. M., DuBois, S. G. and Haas-Kogan, D. A. (2011). Cooperation of the HDAC inhibitor vorinostat and radiation in metastatic neuroblastoma: efficacy and underlying mechanisms. Cancer Lett 306(2): 223-229.
  3. Prasad, G., Sottero, T., Yang, X., Mueller, S., James, C. D., Weiss, W. A., Polley, M. Y., Ozawa, T., Berger, M. S., Aftab, D. T., Prados, M. D. and Haas-Kogan, D. A. (2011). Inhibition of PI3K/mTOR pathways in glioblastoma and implications for combination therapy with temozolomide. Neuro Oncol 13(4): 384-392.
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Yang, X. (2012). Clonogenic Assay. Bio-protocol 2(10): e187. DOI: 10.21769/BioProtoc.187.

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shannon yelbasi
queen's university belfast

I am hoping someone could help me out.
11/22/2015 9:11:27 AM Reply
Maha Soltan
National Research Centre
Please. how could I count the colony's cell number?
1/7/2015 1:26:46 AM Reply
deva umapathy
Bharathidasan University

Dear above friends, could you all see the point of 5th from fixation and staining paragraph, i think it would be wrong one..... is in it....

5/27/2016 7:14:36 AM

Thiago Lima
I have a question regard the well plate used. How can I determine what is the smallest size well can I use for clonogenic survival assays? Is there any where in literature that this has been studied?

Thank you
1/9/2014 5:56:29 AM Reply
fish master
plating before treatment and plating after treatment, what's the difference? which one is usually better?
8/14/2013 4:10:16 PM Reply
Lin Fang
Department of Pediatrics, School of Medicine, Stanford University, USA

Although I have never done such experiment,I'd like to share some of my thought with you. From my understanding of this protocol, it should not affect the experiment fundamentally. Should there is any trivial difference, I prefer plate cell first then treat cells since if the other way (treat cells, then plate them) would involve stress the already stressed (treatment) cells one more time (detach the cells). In addition, for the easiness of operation, it is much easier to plate cell first then the other way around. Since for plate cell first case, you just need to aliquot cells from the same master cell suspension, but if treat cell first, then you need to harvest, count and aliquot from different dish of cells. These are the trivial difference I could think of, but shouldn't change the result.

Hope it may be helpful.

8/25/2013 10:00:37 AM

Could you please tell me about the medium used.
2/22/2013 5:50:21 AM Reply