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Dec 2006
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Clonogenic Assay
克隆形成实验   

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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).

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.

简介

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).

材料和试剂

  1. 细胞培养基
  2. 磷酸盐缓冲盐水(PBS)
  3. 胎牛血清(FBS)
  4. 胰蛋白酶/EDTA(Life Technologies,Invitrogen TM,目录号:25200-056)
  5. 结晶紫(Sigma-Aldrich,目录号:C3886)
  6. 甲醇(Sigma-Aldrich,目录号:34860)
  7. 冰乙酸(Sigma-Aldrich,目录号:320099)
  8. 固定溶液
  9. 菌落固定溶液(见配方)
  10. 水晶紫溶液(见配方)

设备

  1. 细胞培养皿或六孔板(Thermo Fisher Scientific,目录号:08-772-1B)
  2. 血细胞计数器(Hausser Bright-Line)(Thermo Fisher Scientific,目录号:02-671-10)
  3. 立体显微镜(例如,Nikon Eclipse,型号:TS100)
  4. 血细胞计数器
  5. 孵化器

程序

  1. 细胞制备:
    1. 根据需要(例如,GBM细胞系,U87,U251,SF188等)培养细胞。
    2. 取出培养基,然后用10ml PBS冲洗细胞
    3. 加入4毫升0.25%胰蛋白酶的细胞,并在37℃孵育1-5分钟,直到细胞出现圆形。
    4. 加入10毫升含10%FBS的培养基,通过移液分离细胞
    5. 使用血细胞计数器计数细胞 注意:为单元格获取相对准确的数字至关重要。
    6. 准备所需的播种浓度,然后种子细胞到菜或6孔板

  2. 测定设置:
    可以在处理之前或之后对细胞进行电镀。
    1. 治疗前电镀:
      1. 收获细胞并在6孔板上每个培养皿或每孔平板适当数量的细胞,至少一式两份。 用于接种的细胞的数目应该通过处理的侵袭性来确定。 在37℃的CO 2培养箱中孵育细胞数小时,并允许它们附着在平板/皿上。
      2. 根据需要用化学品(例如,,1-100μM),辐射(例如,2-10Gy)或两者的组合处理细胞。
      3. 将细胞在CO 2培养箱中在37℃孵育1-3周,直到对照板中的细胞形成具有基本上良好大小的菌落(每个菌落50个细胞是评分的最小值) 。
    2. 处理后电镀:
      1. 收获细胞后处理。可以电镀50个或高达50×10 4个细胞。准备连续稀释与不同数量的细胞,如果治疗的影响不清楚。对于放射治疗,可以在处理后立即电镀细胞或稍后重新电镀。在重新电镀之前,最好将细胞保存在冰上。
      2. 在CO 2培养箱中在37℃下孵育细胞1-3周,直到对照板中的细胞形成具有基本上良好大小的集落(每个集落50个细胞是评分的最小值)。

  3. 固定和染色:
    1. 取出培养基,然后用10ml PBS冲洗细胞
    2. 取出PBS,加入2-3 ml固定液,并将盘/板在室温(RT)下放置5分钟
    3. 取出固定液。
    4. 加入0.5%结晶紫溶液,在室温下孵育2小时
    5. 加入10毫升含10%FBS的培养基,通过移液分离细胞
    6. 小心地移除结晶紫,将盘/板浸入自来水中冲洗掉结晶紫
    7. 在室温下将餐具/盘子放在桌布上风干几天。

  4. 数据分析:
    1. 用立体显微镜计数菌落数。  
    2. 计算电镀效率(PE)和存活分数(SF) PE = no。 的菌落形成/无。 的细胞接种×100%
      SF = no。 的处理后形成的菌落/的细胞接种x PE

食谱

  1. 菌落固定溶液
    乙酸/甲醇1:7(vol/vol)
  2. 结晶紫0.5%溶液

致谢

此协议已从已发布的版本修改(Franken等人,2006年)。

参考文献

  1. Franken,N.A.,Rodermond,H.M.,Stap,J.,Haveman,J.and van Bree,C.(2006)。 体外细胞克隆形成测定 。 Nat Protoc 1(5):2315-2319。
  2. Mueller,S.,Yang,X.,Sottero,TL,Gragg,A.,Prasad,G.,Polley,MY,Weiss,WA,Matthay,KK,Davidoff,AM,DuBois,SGand Haas-Kogan,DA 2011)。 HDAC抑制剂伏立诺他和放疗在转移性成神经细胞瘤中的合作:功效和基础机制。 Cancer Lett 306(2):223-229。
  3. Prasad,G.,Sottero,T.,Yang,X.,Mueller,S.,James,CD,Weiss,WA,Polley,MY,Ozawa,T.,Berger,MS,Aftab,DT,Prados,MD和Haas -Kogan,DA(2011)。 抑制胶质母细胞瘤中的PI3K/mTOR途径以及与替莫唑胺的联合治疗的意义。 em> Neuro Oncol 13(4):384-392
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Yang, X. (2012). Clonogenic Assay. Bio-protocol 2(10): e187. DOI: 10.21769/BioProtoc.187.
提问与回复
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khem limbu
Khem Raj Limbu
(1) what kind of cell culture medium is suitable for performing this test? could you please describe it.(2) what kind of cytotoxic agents we need to used and can cytotoxic agents affect the cell culture medium?
2021/3/9 0:49:12 回复
Sharavan Ramachandran
Sharavan Ramachandran

Hello Khem,

Please find the answers to your questions below.

1. You have to use a culture medium, where you normally culture your cells. Make sure to monitor the cells periodically because clonogenic assays lasts for more than a week (depends upon the growth kinetics of the cells) and the media might evaporate by that time. So you need to periodically replenish or change fresh media (media change is preferable).

2. You can use any cytotoxic agent of your interest. However, I would recommend to use non/sub-toxic concentrations of your agent because you will seed only 500 - 1000 cells per well in a 6 well plate and because of the cell-drug ratio, toxic conc. of your compound might kill all the cells in a short period of time. Infact, control cells treated with vehicle (same volume as your treatment) might not form colonies. The results will then be inconclusive with loose ends.

2021/3/9 1:15:21 回复


khem limbu
Khem Raj Limbu

Thank you for giving such a great information.

2021/3/11 17:38:29 回复


Sam Marchant
Sam Willard Marchant
Should you plate the same number of cells for all treatment concentrations and PE controls. What is the general amount plated for an immortalized cell line being treated with chemo-drug in a 6 well plate?
2021/3/5 14:22:36 回复
Sharavan Ramachandran
Sharavan Ramachandran

Hello Sam,

You should seed the same number of cells across all the groups. You can seed 500 cells per well in a 6 well plate. For more information, please refer to my answer to Khem's question.

2021/3/9 1:16:54 回复


shannon yelbasi
queen's university belfast
Hello,

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

2016/5/27 7:14:36 回复


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
2014/1/9 5:56:29 回复
fish master
genetics
plating before treatment and plating after treatment, what's the difference? which one is usually better?
2013/8/14 16:10:16 回复
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.

2013/8/25 10:00:37 回复


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