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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).

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Clonogenic Assay
克隆形成实验

癌症生物学 > 细胞死亡 > 细胞生物学试验 > 细胞活性
作者: Xiaodong Yang
Xiaodong YangAffiliation: Department of Neurology, University of California, San Francisco, USA
For correspondence: yangxiaodong1@yahoo.com
Bio-protocol author page: a43
Vol 2, Iss 10, 5/20/2012, 36740 views, 5 Q&A
DOI: https://doi.org/10.21769/BioProtoc.187

[Abstract] 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).

[Abstract] 克隆生成测定作为一个有用的工具用于检测在肿瘤治疗后是否减少了肿瘤细胞克隆存活率,一个群体并定义为由至少50个细胞形成的簇,这个可以在显微镜下测定 。克隆生成测定是一种测定细胞在电离辐射处理下的生殖死亡方法,也可以用于测定其它的细胞毒剂的效应。, 下面的方法是发表版本的改进版(Franken, Rodermond 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)

Equipment

  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

Procedure

  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

Recipes

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

Acknowledgments

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

References

  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.

材料和试剂

 

1.         细胞培养基

2.         PBS

3.         FBS

4.         胰蛋白胨/ EDTA (Invitrogen, 25200056)

5.         结晶紫(Sigma-Aldrich, C3886)

6.         甲醇 (Sigma-Aldrich, 34860)

7.         冰醋酸(Sigma-Aldrich, 320099)

8.         ddH2O

 

仪器

 

1.      细胞培养皿或者六孔盘(Fisher Scientific, 08-772-1B)

2.      细胞计数器 (Hausser Bright-Line, Fisher Scientific, 02-671-10)

3.      体视显微镜(e.g., Nikon Eclipse TS100)

 

步骤

 

1.         细胞制备

1)      根据需求培养细胞(e.g., GBM cell lines, U87, U251, SF188, etc). 

2)      去掉培养基,用10ml PBS洗细胞。 

3)      向细胞中加4ml 0.25%胰蛋白酶在 37 °C 下孵育1-5min直到细胞变成圆形。

4)      加入 10ml  含有10% FBS的培养基, 用移液器分离细胞. 

5)      用细胞计数器数细胞。 

注意: 细胞的数目必须精确,这个很重要。

6)      准备想要的接种浓度,并且接种细胞到培养皿或6孔皿中。

2.         检测步骤:

细胞可以在处理前后接种。. 

1)      处理前接种  

a.      收集细胞,接种适当数目的细胞到培养皿或者6孔盘中,至少两份。根据处理的强弱来确定接种细胞数量。在37°C的二氧化碳孵化器中孵育几个小时使其贴壁。. 

b.      根据需要用化学品(e.g., 1-100 μM),放射(e.g., 2-10 Gy)或则两者联合处理细胞。

c.      37°C的二氧化碳孵化器中孵育1-3 星期直到细胞大量的形成群体。 (最少每个群体有50 细胞). 

2)      处理后接种 

a.      处理后收集细胞, 50 或者直到 50x104 可以被接种,准备被连续稀释不同数量的细胞,因为处理的效果不能确定。对于放射处理,细胞可以在被处理后迅速接种或者稍后接种。最好在重新接种前将细胞保持在冰上。

b.      37°C的二氧化碳孵化器中孵育1-3 星期直到细胞大量的形成群体。 (最少每个群体有50 细胞).. 

3.         固定和染色:

1)      去掉培养基,用10ml PBS洗细胞. 

2)      去掉PBS2-3ml 固定溶液使其在室温下5分钟。

3)      移去固定液。 

4)      加入0.5% 结晶紫溶液并且在室温下孵育2小时。

5)      加入 10ml  含有10% FBS的培养基, 用移液器分离细胞.. 

6)      小心的移去结晶紫溶液,并且将盘子浸入流动的水中出去结晶紫。

7)      在室温下放置几天风干盘子 

4.         数据分析:

1)      在体视显微镜下数群体数量。 

2)      计算接种效率(PE)和存活数 (SF).

PE = 群体形成数量/ 细胞接种数量 X 100%

SF = 处理后的群体数量/细胞接种数量X PE

 

配置方法

 

1.        Colony fixation solutions

Acetic acid/methanol 1:7 (vol/vol)

2.        Crystal violet, 0.5% solution

 

摘要

 

1.        Franken N.A., Rodermond H.M., Stap J., Haveman J., van Bree C. (2006). Clonogenic assay of cells in vitro. Nat Protoc 1(5): 2315-9. 

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., Haas-Kogan D.A. (2011). Cooperation of the HDAC inhibitor vorinostat and radiation in metastatic neuroblastoma: efficacy and underlying mechanisms. Cancer Letters 306(2): 223-9. 

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., Haas-Kogan D.A. (2011). Inhibition of PI3K/mTOR pathways in glioblastoma and implications for combination therapy with temozolomide. Neuro Oncol 13(4): 384-92. 

 

 

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How to cite this protocol: Yang, X. (2012). Clonogenic Assay. Bio-protocol 2(10): e187. DOI: 10.21769/BioProtoc.187; Full Text



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1/7/2015 1:26:46 AM  

Maha Soltan
National Research Centre

Please. how could I count the colony's cell number?

5/27/2016 7:14:36 AM  

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....

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1/9/2014 5:56:29 AM  

Thiago Lima
UCL

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
Thiago

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8/14/2013 4:10:16 PM  

fish master
genetics

plating before treatment and plating after treatment, what's the difference? which one is usually better?

8/25/2013 10:00:37 AM  

Lin Fang
Stanford University

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.

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2/22/2013 5:50:21 AM  

Could you please tell me about the medium used.

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