欢迎您, 登录 | 注册

首页 | English

X
加载中

The Comet assay (or Single Cell Gel Electrophoresis assay) is a sensitive technique to detect DNA damage at the level of an individual cell. This technique is based on micro-electrophoresis of cells DNA content. Briefly, cells are embedded in agarose, lysed and submitted to an electric field, before the staining step with a fluorescent DNA binding dye. Damaged DNA (charged DNA) migrates in this field, forming the tail of a “comet”, while undamaged DNA remained in the head of the “comet”. The following document describes the protocol to realize a neutral comet assay. This assay can be applied to different cell types and has been useful for numerous applications in fields of toxicology or DNA damage and repair.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Neutral Comet Assay
中性单细胞凝胶电泳分析

癌症生物学 > 通用技术 > 生物化学试验 > DNA结构和改变
作者: Elisa Boutet-Robinet
Elisa Boutet-RobinetAffiliation: Toxalim, Research Centre in Food Toxicology, INRA, Université de Toulouse, UPS, UMR1331, Toulouse, France
For correspondence: elisa.boutet@univ-tlse3.fr
Bio-protocol author page: a854
Didier Trouche
Didier TroucheAffiliation: LBCMCP CNRS UMR5088, University of Toulouse UPS, Toulouse, France
Bio-protocol author page: a853
 and Yvan Canitrot
Yvan CanitrotAffiliation: LBCMCP CNRS UMR5088, University of Toulouse UPS, Toulouse, France
Bio-protocol author page: a451
Vol 3, Iss 18, 9/20/2013, 5287 views, 1 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.915

[Abstract] The Comet assay (or Single Cell Gel Electrophoresis assay) is a sensitive technique to detect DNA damage at the level of an individual cell. This technique is based on micro-electrophoresis of cells DNA content. Briefly, cells are embedded in agarose, lysed and submitted to an electric field, before the staining step with a fluorescent DNA binding dye. Damaged DNA (charged DNA) migrates in this field, forming the tail of a “comet”, while undamaged DNA remained in the head of the “comet”. The following document describes the protocol to realize a neutral comet assay. This assay can be applied to different cell types and has been useful for numerous applications in fields of toxicology or DNA damage and repair.

Keywords: Comet assay(彗星实验), Genotoxicity(genotoxicity), DNA damage(DNA损伤)

Materials and Reagents

  1. Cells to analyze
  2. Low Melting Point (LMP) Agarose (Sigma-Aldrich, catalog number: A9414)
  3. Seakem® Agarose (Ozyme, catalog number: LON50004)
  4. PBS (Ca2+ and Mg2+-free phosphate-buffered saline)
  5. 5 N NaOH
  6. 0.5 M EDTA disodium salt solution (pH 8)
  7. Trisma base
  8. Triton X-100
  9. N-Lauroylsarcosine (Sigma-Aldrich, catalog number: L5125)
  10. Dimethylsulphoxide (DMSO)
  11. Absolute ethanol
  12. Ethidium bromide (10 mg/ml)
  13. Trypsin/EDTA
  14. Sodium acetate
  15. Lysis solution (see Recipes)
  16. Electrophoresis solution (see Recipes)

Equipment

  1. Microscope Super Frost plus glass slides
  2. Malassez chamber
  3. Microscope coverslips (22 x 22 mm)
  4. Microscope coverslips (24 x 32 mm)
  5. Centrifuges
  6. Electrophoresis tank: Econo-Submarine (20 cm x 30 cm) (C.B.S. Scientific, USA)
  7. Fluorescence microscope, camera and software (e.g. Nikon Eclipse 50i microscope equipped with a Luca S camera and Komet 6.0 software)

Software

  1. Komet 6.0 software (Andor Technology)

Procedure

  1. Prepare agarose solution and slides.
    1. At least 24 h before the experiments:
      1. Prepare 0.8% solution of Seakem® Agarose in PBS.
      2. Pre-coat Super Frost slides by dipping in a vertical jar containing melted agarose, stirred with a magnetic stirrer and kept at 100 °C (Figure 1).


        Figure 1. Coating of the slides with agarose

      3. Drain off the agarose in excess by wiping the back of the slides (Figure 2).


        Figure 2. Slides coating with agarose. Removing of the agarose on the back of the slides.

      4. Let the slides dry and then store at room temperature until use.
    2. At least 2 h before the experiments: prepare 0.7% solution of LMP agarose in PBS and place it at 37 °C in a water-bath until use.
  2. Prepare cells
    The number of harvested cells can be adjusted according to the size of the cells. Cells could be numbered either by an automated cell counter or a counting chamber (e.g. Malassez chamber).
    If cells used are adherent, cells must be carefully detached with trypsin/EDTA and isolated before centrifugation and further use.
  3. Embed cells in LMP agarose (in a dark room):
    1. After cell centrifugation, discard the supernatant and resuspend the pellet of cells (150,000 to 200,000 cells) by gently pipetting with 200 μl of 0.7% LMP agarose.
    2. Lay 65 μl of agarose containing the cells on each pre-coated glass slide.
    3. Immediately cover with a 24 x 32 mm coverslip.
    4. Put the slide on an ice-pack for solidification during 5-10 minutes.
    5. Slide off the coverslip to remove it.
    6. Finally cover with 80 μl of LMP agarose (top agarose layer) and cover again with a 24 x 32 mm coverslip.
    7. Put the slide again on an ice-pack for solidification during 5-10 minutes.
    8. Remove the coverslip.
  4. Lysis and electrophoresis (in a dark room):
    1. Place the slides in lysis solution for at least 1 h at 4 °C.
    2. Wash three times for 5 min with the electrophoresis buffer.
    3. Transfer the slides in the electrophoresis tank filled with electrophoresis solution (Figure 3).


      Figure 3. Electrophoresis of the slides. Disposition of the slides in the tank. In this case eight slides are subjected to electrophoresis, four by row.

    4. Proceed to electrophoresis at 18 V (0.5 V/cm) during 1 h.
    5. Wash in PBS for 2 x 5 min.
  5. Dehydration, staining and analysis:
    1. Fix the cells with 2 x 10 min washes in absolute ethanol, air-dry for at least 2 hours at room temperature.
    2. Add 50 μl ethidium bromide (2 μg/ml in water) on the microscope slide and cover with a 22 x 22 mm coverslip for staining.
    3. Analyze the cells: score 50 cells per slide, 2 slides per condition with the fluorescence microscope equipped with a camera and adapted software (Figures 4 and 5).


      Figure 4. Representing analysis of each cell

      a = head length
      b = tail length
      % tail DNA = fraction of DNA in the tail
      Tail moment = % tail DNA x b
      Comet tail length can be calculated by different ways depending on the authors. Tail moment is a common parameter used to characterize the comet. For this, the fraction of DNA in the tail is evaluated by the fluorescence in the tail and divided by the total fluorescence (in the head and in the tail) to be expressed in percentage. Tail moment is the product of % tail DNA and tail length.


      Figure 5. Images representing nucleus of undamaged cells, negative for the presence of comet (left panel) and nucleus of damaged cells presenting comet (right panel)

      As a positive control, cells irradiated with ionizing radiations at 20 Gy and examined just after irradiation are a good control as shown in the photographs above.

Recipes

  1. Lysis solution
    2.5 M NaCl
    0.1 M EDTA
    10 mM Trizma base (pH 10)
    1% N-laurylsarcosine
    0.5% Triton X-100
    10% DMSO final
    Keep at 4 °C
  2. Electrophoresis solution
    300 mM sodium acetate
    100 mM Tris-HCl (pH 8.3) at 4 °C

Acknowledgments

This protocol was adapted from previously published papers (Courilleau et al., 2012; Olive et al., 1990; Ostling and Johanson,  1984; Ostling and Johanson, 1987; Wojewodzka et al., 2002).

References

  1. Courilleau, C., Chailleux, C., Jauneau, A., Grimal, F., Briois, S., Boutet-Robinet, E., Boudsocq, F., Trouche, D. and Canitrot, Y. (2012). The chromatin remodeler p400 ATPase facilitates Rad51-mediated repair of DNA double-strand breaks. J Cell Biol 199(7): 1067-1081. 
  2. Olive, D. M., Johny, M. and Sethi, S. K. (1990). Use of an alkaline phosphatase-labeled synthetic oligonucleotide probe for detection of Campylobacter jejuni and Campylobacter coli. J Clin Microbiol 28(7): 1565-1569.
  3. Ostling, O. and Johanson, K. J. (1984). Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123(1): 291-298.
  4. Ostling, O. and Johanson, K. J. (1987). Bleomycin, in contrast to gamma irradiation, induces extreme variation of DNA strand breakage from cell to cell. Int J Radiat Biol Relat Stud Phys Chem Med 52(5): 683-691.
  5. Wojewodzka, M., Buraczewska, I. and Kruszewski, M. (2002). A modified neutral comet assay: elimination of lysis at high temperature and validation of the assay with anti-single-stranded DNA antibody. Mutat Res 518(1): 9-20.


How to cite this protocol: Boutet-Robinet, E., Trouche, D. and Canitrot, Y. (2013). Neutral Comet Assay. Bio-protocol 3(18): e915. DOI: 10.21769/BioProtoc.915; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
11/17/2014 7:01:57 AM  

nive thiagarajan
psg college of technology

gel is coming out of the slide during lysing and electrophoresis. how to make it stick to the slide?

11/24/2014 9:10:00 AM  

Elisa Boutet-Robinet (Author)
Toxalim, Research Centre in Food Toxicology, INRA, Université de Toulouse, UPS, UMR1331, France

There are different critical steps about the gel (especially the points 3.2 to 3.8 of the on-line protocol) that can influence how it will stick during all the experiment. The first important thing is the temperature of the slide and of the buffers. Everything must be cold. The room temperature must also not be too high. The time (5 to 10 minutes) for which the slide stay on ice-pack and the temperature of the ice-pack is also important (take it out of the freezer 10-20 minutes before the use for the experiment). It is also important to slide off very gently the coverslip after the agarose has solidified.

12/2/2014 5:11:55 AM  

nive thiagarajan
psg college of technology

Thank you so much. The cold temperature tip really helped :)

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook
Didier Trouche的其他实验方案(1)
Yvan Canitrot的其他实验方案(1)