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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.

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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, 6927 views, 1 Q&A
DOI: https://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)

[Abstract]

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.

材料和试剂

  1. 要分析的单元格
  2. 低熔点(LMP)琼脂糖(Sigma-Aldrich,目录号:A9414)
  3. Seakem 琼脂糖(Ozyme,目录号:LON50004)
  4. PBS(Ca 2+和Mg 2+ +无磷酸盐缓冲盐水)中的至少一种。
  5. 5 N NaOH
  6. 0.5 M EDTA二钠盐溶液(pH 8)
  7. Trisma碱基
  8. Triton X-100
  9. N-月桂酰肌氨酸(Sigma-Aldrich,目录号:L5125)
  10. 二甲基亚砜(DMSO)
  11. 绝对乙醇
  12. 溴化乙锭(10mg/ml)
  13. 胰蛋白酶/EDTA
  14. 醋酸钠
  15. 裂解溶液(参见配方)
  16. 电泳溶液(参见配方)

设备

  1. 显微镜超级霜和玻璃滑块
  2. Malassez房间
  3. 显微镜盖玻片(22×22mm)
  4. 显微镜盖玻片(24×32mm)
  5. 离心机
  6. 电泳槽:经济潜水艇(20cm x 30cm)(美国科学科学院)
  7. 荧光显微镜,照相机和软件(例如配备有Luca S照相机和Komet 6.0软件的Nikon Eclipse 50i显微镜)

软件

  1. Komet 6.0软件(安卓技术)

程序

  1. 准备琼脂糖溶液和幻灯片。
    1. 实验前至少24小时:
      1. 制备0.8%的Seakem 琼脂在PBS中的溶液
      2. 通过浸渍在含有熔化的琼脂糖的垂直罐中预涂覆超级霜冻载玻片,用磁力搅拌器搅拌并保持在100℃(图1)。


        图1.用琼脂糖涂布载玻片

      3. 通过擦拭载玻片的背面,过量地除去琼脂糖(图2)。


        图2.涂有琼脂糖的玻片。取出载玻片背面的琼脂糖。

      4. 让载玻片干燥,然后在室温下储存,直到使用。
    2. 在实验前至少2小时:制备0.7%的LMP琼脂糖的PBS溶液,并将其置于37℃水浴中直至使用。
  2. 准备单元格
    可以根据细胞的大小调整收获的细胞的数量。 可以通过自动细胞计数器或计数室(例如Malassez室)对细胞进行编号。
    如果使用的细胞是粘附的,细胞必须小心地用胰蛋白酶/EDTA分离,并在离心和进一步使用之前分离
  3. 将细胞嵌入LMP琼脂糖(在暗室中):
    1. 细胞离心后,弃去上清液,用200μl的0.7%LMP琼脂糖轻轻吹打重悬细胞沉淀(150,000至200,000个细胞)。
    2. 将65微升含有细胞的琼脂糖置于每个预涂玻片上
    3. 立即盖上一个24 x 32毫米的盖玻片。
    4. 将载玻片放在冰袋上固化5-10分钟。
    5. 滑动盖玻片以将其移除。
    6. 最后盖上80微升的LMP琼脂糖(顶层琼脂糖层),再盖上一个24×32毫米的盖玻片。
    7. 将载玻片再次放在冰袋上固化5-10分钟。
    8. 取出盖玻片。
  4. 裂解和电泳(在暗室中):
    1. 将载玻片放在裂解液中至少1小时,在4℃
    2. 用电泳缓冲液洗涤三次,每次5分钟。
    3. 在装有电泳溶液的电泳槽中转移载玻片(图3)

      图3.载玻片的电泳。在槽中处理载玻片。 在这种情况下,8个载玻片进行电泳,每行4个
    4. 在18 V(0.5 V/cm)下进行电泳1小时。
    5. 在PBS中洗2次,每次5分钟。
  5. 脱水,染色和分析:
    1. 固定细胞在无水乙醇中2×10分钟洗涤,在室温下风干至少2小时
    2. 在显微镜载玻片上加入50微升溴化乙锭(2微克/毫升,在水中),盖上一个22 x 22毫米的盖玻片进行染色。
    3. 分析细胞:每张幻灯片评分50个细胞,每个条件2个载玻片,配备有照相机和适配软件的荧光显微镜(图4和5)。


      图4.表示每个单元格的分析

      a =头长
      b =尾长
      %尾DNA =尾巴中DNA的比例
      尾矩=%尾DNA×b
      彗尾长度可以通过不同的方法计算,取决于作者。尾矩是用于表征彗星的常用参数。为此,通过尾部中的荧光评估尾部中的DNA分数,并除以以百分比表示的总荧光(在头部和尾部中)。尾矩是%尾DNA和尾长的乘积

      图5.表示未受损细胞核的图像,对于存在彗星(左图)和呈现彗星的受损细胞的细胞核(右图)为阴性的图像

      作为阳性对照,在20Gy下用电离辐射照射并在照射后立即检查的细胞是如上述照片中所示的良好对照。

食谱

  1. 裂解液
    2.5 M NaCl
    0.1 M EDTA
    10mM Trizma碱(pH 10)
    1%N-月桂基肌氨酸 0.5%Triton X-100 10%DMSO最终
    保持在4°C
  2. 电泳溶液
    300mM乙酸钠 100mM Tris-HCl(pH8.3),4℃

致谢

该方案改编自以前发表的论文(Courilleau等人,2012; Olive等人,1990; Ostling和Johanson,1984; Ostling和Johanson,1987) ; Wojewodzka等人,2002)。

参考文献

  1. Courilleau,C.,Chailleux,C.,Jauneau,A.,Grimal,F.,Briois,S.,Boutet-Robinet,E.,Boudsocq,F.,Trouche,D。和Canitrot, 染色质重塑p400 ATP酶促进Rad51介导的DNA双链断裂修复。 J Cell Biol 199(7):1067-1081。
  2. Olive,D.M.,Johny,M。和Sethi,S.K。(1990)。 使用碱性磷酸酶标记的合成寡核苷酸探针检测空肠弯曲杆菌 >和弯曲杆菌。 J Clin Microbiol 28(7):1565-1569。
  3. Ostling,O。和Johanson,K.J。(1984)。 辐射诱导的DNA损伤在个体哺乳动物细胞中的微电泳研究生化Biophys Res Commun 123(1):291-298
  4. Ostling,O。和Johanson,K.J。(1987)。 与γ-照射相反,博来霉素诱导DNA链从细胞到细胞的破坏的极端变异。/a> Int J Radiat Biol Relat Stud Phys Chem Med 52(5):683-691。
  5. Wojewodzka,M.,Buraczewska,I。和Kruszewski,M。(2002)。 经过修改的中性彗星测定:消除高温下的裂解并验证抗单克隆抗体-stranded DNA antibody。 518(1):9-20。
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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



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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 :)

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