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Detection of Anaphase Bridge Formation by Immunofluorescence Microscopy in Mammalian Cells
采用免疫荧光显微技术检测哺乳动物细胞分裂后期桥的形成   

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Abstract

The aim of this protocol is to provide a comprehensive description of the materials, equipment and reproducible methods to detect and analyze anaphase bridges in immunofluorescence microscopy using DAPI to detect cells that failed to completely segregate during mitosis. It describes the process of cell preparation, staining and microscopic settings for detection of anaphase bridges. The protocol has been adapted from our previous publication (Aschacher et al., 2016).

Keywords: Anaphase bridges(后期桥), Cancer(癌症), DNA Damage(DNA损伤)

Background

During cell division it is vital for the maintenance of genome integrity that the genetic material is fully separated. For various reasons this process can be dysfunctional and as a result the sister chromatids are connected by DNA bridges, which most frequently happens during anaphase. Especially chromosomal fragile sites are associated with anaphase bridges (e.g., unprotected and unstable telomeres). Breakage, deletion, translocation non-disjunction and changes in chromosome number at these sites are often linked with cancer and other genetic diseases. Two types of anaphase bridges are described, the ultrafine DNA bridges, that cannot be detected by DAPI staining and the chromatin bridges, which are visualized by DAPI (Germann et al., 2014). The latter is described subsequently.

This protocol describes a fast and simple method for the detection and calculation of anaphase bridges to provide an additional assay for telomere attrition in any publications.

Materials and Reagents

  1. 8-chamber slides (e.g., NuncTM Lab-TekTM II chamber SlideTM system, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 154534 )
  2. Nail polish (colourless, commercial available)
  3. Coverslips (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: Q10143263NR1 )
  4. Tumour cell line
    Note: HCT-116 p53/wt was used in this protocol (ATCC, Germany).
  5. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 26140079 )
  6. Dulbecco’s phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: D8662 )
  7. Trypsin-EDTA solution, 0.25%, sterile-filtered (Sigma-Aldrich, catalog number: T4049 )
  8. McCoy’s 5A medium modified with sodium bicarbonate, without L-glutamine (Sigma-Aldrich, catalog number: M8403 )
  9. Formaldehyde solution (37%) (Sigma-Aldrich, catalog number: 252549 )
  10. Nuclear staining reagent (colour depends on secondary staining)
    1. 4’,6-diamidino-2-phenylindole (DAPI), colour blue (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 62247 )
    2. Hoechst 33342, trihydrochloride, trihydrate, colour blue (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: H3570 )
    3. DRAQ5TM fluorescent probe solution (5 mM), colour red (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 62251 )
    4. Propidium iodide (PI), colour red (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: P21493 )
  11. Vectashield mounting medium (Vector Laboratories, USA)
  12. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  13. Growth medium (see Recipes)
  14. 3.7% formaldehyde solution (see Recipes)
  15. Permeabilization solution (see Recipes)

Equipment

  1. Cell culture microscope (e.g., Carl Zeiss, Vienna, Austria)
  2. Pipettes
  3. Hemocytometer (e.g., Bürker-Türk) (BRAND, catalog number: 719505 )
  4. Centrifuge (e.g., Eppendorf, Germany)
  5. 37 °C, 5% CO2 incubator
  6. Confocal laser scanning microscope (Carl Zeiss, model: LSM 800 )

Software

  1. Software for LSM800: ZEN lite (for free download find 'www.zeiss.com/microscopy/en_de/products/microscope-software/zen-lite.html')

Procedure

  1. Maintain cells in McCoy’s 5A medium supplemented with 10% fetal bovine serum in a cell culture plate until cells are 70-80% confluent.
  2. Wash cells once with PBS and trypsinize cells by adding 1 ml trypsin solution and incubate for 3-5 min until cells are floating under microscopy.
  3. Add 5-10 ml growth medium, mix them with a pipette, and count cells with a hemocytometer.
  4. Seed cells on chamber slides with appropriate seeding density. The number of cells for a confluent chamber slide will vary with cell type. For this experiment, we use HCT-116 with a seeding density of 2 x 104 cells/well in a 4-chamber slide.
  5. Allow to rest for 24 h or treat the cells as required for 24-72 h in an incubator 37 °C 5% CO2.
  6. Prepare formaldehyde solution with 1 ml 37% formaldehyde in 9 ml 1x PBS, giving a final concentration of formaldehyde of 3.7%. This solution should be prepared immediately before use.
    Note: Formaldehyde is toxic. Please read the MSDS before working with this chemical. Gloves and safety glasses should be worn and solutions made inside a fume hood.
  7. Next day, or after treatment, wash cells twice with ice-cold PBS and fix them with 200-300 µl 3.7% formaldehyde solution for 10 min on ice.
  8. Afterwards, wash the cells twice with PBS for 5 min each at RT (room temperature).
  9. Permeabilize cells by covering cells with permeabilization solution for 10 min at RT.
  10. Wash cells twice with PBS.
  11. Optional: subsequently, proceed to block cells with blocking reagent and stain cells with antibodies.
  12. Afterwards, remove supernatant and stain cells with either DAPI, PI, Hoechst 33342 or DRAQ5 with a final concentration of 1 µg/ml for 15-30 min, respectively in the dark at RT.
    Notes:
    1. Blue spectral properties make it ideal for use with green (Alexa Fluor® 488, FITC, GFP) and red (Alexa Fluor® 594, rhodamine, Texas Red®, mCherry, mKate-2) fluorophores in multicolor experiments. For live-cell staining, Hoechst® 33342 dye is a popular cell-permeant nuclear counterstain.
    2. Optional: Use vectashield mounting medium including 0.5 µg/ml DAPI (Vector Laboratories, USA) the nuclear staining step is not required. Proceed with step 15.
  13. Rinse with PBS and carefully remove the chambers from slides.
  14. Allow the slides to dry for 2-3 min.
  15. Mount coverslip with a drop of mounting medium.
  16. Seal coverslip with nail polish to prevent drying and movement under microscope.
  17. Use a Zeiss LSM (Laser Scanning Microscope) 700 META microscopy laser system, and a ZEN 2010 microscopy system equipped with a 63x and/or 100x objective.
  18. Allow the microscope and lasers to warm up for 15-20 min before use.
  19. The setting used for stain depends on nuclear staining:
    1. DAPI (Laserline: 405) excitation/emission 350/470 (nm);
    2. Propidium iodide excitation/emission 535/617 (nm);
    3. Hoechst 33342 excitation/emission 350/461 (nm);
    4. DRAQ5 excitation/emission 647/681 (nm).
  20. First use 63x magnification objective to find nuclei and capture a minimum of 500 nuclei. For better resolution of anaphase bridges a 100x objective could be used.
  21. For statistical analyses count cell nuclei in a) mitosis (M), b) anaphases (A), and c) anaphases with bridges (AB) separately (Figures 1a and 1b).


    Figure 1. Fluorescence determination of tumor cells with anaphase-bridges. a. Cells in mitosis (M) and specific anaphase with anaphase-bridge (AB) are shown. Scale bar = 10 µm. b. A clear difference could be identified by enlargement of these nuclei in specific cell cycle phase. Representative images of cell nuclei in different cell cycle phase are shown. A. Interphase, B. Prometaphase, C. Metaphase, D. Anaphase, and E. Anaphase with bridge. Scale bars = 5 µm. All cells were stained with DAPI staining. The 'bridge' of anaphase bridges is marked (white arrow).

Data analysis

The frequency of anaphase bridges is calculated as the ratio between cells exhibiting anaphase bridges and the total number of anaphase cells counting at least 100 anaphases. For example we distinguish for calculation i) all nuclei in all phases, ii) all cells in mitosis, iii) cells in anaphases, and iv) anaphase with bridges (also please find Figure 1b). For statistical significance Student's t-test values are calculated.
Note: For this protocol, we treated tumour cell lines with scrambled siRNA or effective siRNA against LINE-1 mRNA as described previously (Aschacher et al., 2016). An effective LINE-1 siRNA treatment causes telomere attrition within three days and leads to anaphase bridges (Table 1 and Figure 2).

Table 1. Statistical calculation for anaphase bridges. Representative table for counting of anaphase bridges is given. A minimum of three independent experiments should be done.



Figure 2. Graphic example for statistical visualization of anaphase bridges. Counted nuclei (including interphase and mitosis), mitosis, anaphases and cells with anaphase bridges are given separately. Numbers of each count are given on the top of bars.

Recipes

  1. Growth medium
    500 ml McCoy’s 5A medium
    50 ml FBS, heat inactivated
  2. 3.7% formaldehyde solution
    1 ml 37% formaldehyde
    9 ml 1x PBS
  3. Permeabilization solution
    1x PBS containing 0.25% Triton X-100

Acknowledgments

This protocol was mainly adapted from the publication Aschacher et al. (2016). The work was supported by the Medical University of Vienna in the laboratories for surgical research.

References

  1. Aschacher, T., Wolf, B., Enzmann, F., Kienzl, P., Messner, B., Sampl, S., Svoboda, M., Mechtcheriakova, D., Holzmann, K. and Bergmann, M. (2016). LINE-1 induces hTERT and ensures telomere maintenance in tumour cell lines. Oncogene 35(1): 94-104.
  2. Germann, S. M., Schramke, V., Pedersen, R. T., Gallina, I., Eckert-Boulet, N., Oestergaard, V. H. and Lisby, M. (2014). TopBP1/Dpb11 binds DNA anaphase bridges to prevent genome instability. J Cell Biol 204(1): 45-59.

简介

The aim of this protocol is to provide a comprehensive description of the materials, equipment and reproducible methods to detect and analyze anaphase bridges in immunofluorescence microscopy using DAPI to detect cells that failed to completely segregate during mitosis. It describes the process of cell preparation, staining and microscopic settings for detection of anaphase bridges. The protocol has been adapted from our previous publication (Aschacher et al., 2016).

[Background] During cell division it is vital for the maintenance of genome integrity that the genetic material is fully separated. For various reasons this process can be dysfunctional and as a result the sister chromatids are connected by DNA bridges, which most frequently happens during anaphase. Especially chromosomal fragile sites are associated with anaphase bridges (e.g., unprotected and unstable telomeres). Breakage, deletion, translocation non-disjunction and changes in chromosome number at these sites are often linked with cancer and other genetic diseases. Two types of anaphase bridges are described, the ultrafine DNA bridges, that cannot be detected by DAPI staining and the chromatin bridges, which are visualized by DAPI (Germann et al., 2014). The latter is described subsequently.
   This protocol describes a fast and simple method for the detection and calculation of anaphase bridges to provide an additional assay for telomere attrition in any publications.

关键字:后期桥, 癌症, DNA损伤

材料和试剂

  1. 8孔玻片(例如,,, TM Lab-Tek Scientific,Thermo Scientific TM ,目录号:154534)
  2. 指甲油(无色,市售)
  3. 盖玻片(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:Q10143263NR1)
  4. 肿瘤细胞系
    注意:在本方案(ATCC,德国)中使用HCT-116p53/wt。
  5. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:26140079)
  6. Dulbeccos的磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:D8662)
  7. 胰蛋白酶-EDTA溶液,0.25%,无菌过滤(Sigma-Aldrich,目录号:T4049)
  8. 用碳酸氢钠改性的不含L-谷氨酰胺的McCoy's 5A培养基(Sigma-Aldrich,目录号:M8403)
  9. 甲醛溶液(37%)(Sigma-Aldrich,目录号:252549)
  10. 核染色试剂(颜色取决于二次染色)
    1. 4',6-二脒基-2-苯基吲哚(DAPI),蓝色(Thermo Fisher Scientific,Thermo Scientific TM,目录号:62247)
    2. Hoechst 33342,三盐酸盐,三水合物,蓝色(Thermo Fisher Scientific,Thermo Scientific ,目录号:H3570)
    3. DRAQ5 TM荧光探针溶液(5mM),红色(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:62251)
    4. 碘化丙锭(PI),红色(Thermo Fisher Scientific,Molecular Probes ,目录号:P21493)
  11. Vectashield封固剂(Vector Laboratories,USA)
  12. Triton X-100(Sigma-Aldrich,目录号:X100)
  13. 生长培养基(见Recieps)
  14. 3.7%甲醛溶液(见配方)
  15. 渗透溶液(参见配方)

设备

  1. 细胞培养显微镜(例如,Carl Zeiss,Vienna,Austria)
  2. 移液器
  3. 血细胞计数器(例如,Bürker-Türk)(BRAND,目录号:719505)
  4. 离心机(例如,Eppendorf,德国)
  5. 37℃,5%CO 2培养箱
  6. 共聚焦激光扫描显微镜(Carl Zeiss,型号:LSM 800)

软件

  1. LSM800的软件:ZEN lite(免费下载,查找' www.zeiss.com/microscopy/en_de/products/microscope-software/zen-lite.html ')

程序

  1. 在细胞培养板中维持细胞在补充有10%胎牛血清的McCoy's 5A培养基中,直到细胞70-80%汇合。
  2. 用PBS洗涤细胞一次,通过加入1ml胰蛋白酶溶液胰蛋白酶处理细胞,孵育3-5分钟,直到细胞在显微镜下漂浮。
  3. 加入5-10 ml生长培养基,用移液管混合,用血细胞计数器计数细胞
  4. 种子细胞在室滑片上具有合适的接种密度。用于汇合室载玻片的细胞数目将随细胞类型而变化。对于该实验,我们使用在4室载玻片中具有2×10 4个细胞/孔的接种密度的HCT-116。
  5. 允许休息24小时或根据需要在37℃,5%CO 2的培养箱中处理细胞24-72小时。
  6. 用1ml 37%甲醛在9ml1×PBS中制备甲醛溶液,得到3.7%的甲醛终浓度。该溶液应在使用前立即制备。
    注意:甲醛是有毒的。使用本化学品前请阅读MSDS。应佩戴手套和安全眼镜,并在通风橱内制作解决方案。
  7. 第二天或治疗后,用冰冷的PBS洗涤细胞两次,并用200-300μl3.7%甲醛溶液在冰上固定10分钟。
  8. 然后,用PBS洗涤细胞两次,每次5分钟,室温(室温)
  9. 通过用透化溶液在室温覆盖细胞10分钟来透化细胞
  10. 用PBS洗涤细胞两次。
  11. 任选:随后,用阻断剂进行阻断细胞,用抗体染色细胞
  12. 然后,在暗处,在RT下,用DAPI,PI,Hoechst 33342或DRAQ5分别除去上清液和染色细胞,最终浓度为1μg/ml,持续15-30分钟。
    注意:
    1. 488,FITC,GFP)和红色(Alexa Fluor mCherry,mKate-2)荧光团。对于活细胞染色,Hoechst 33342染料是一种流行的细胞渗透性核复染剂。
    2. 可选:使用包括0.5μg/ml DAPI(Vector Laboraties,USA)的vectashield封固剂,不需要核染色步骤。继续执行第15步。
  13. 用PBS冲洗,小心地从幻灯片中取出试剂盒。
  14. 让幻灯片干燥2-3分钟。
  15. 用一滴安装介质安装盖玻片。
  16. 密封盖子用指甲油,以防止干燥和显微镜下的移动
  17. 使用Zeiss LSM(激光扫描显微镜)700 META显微镜激光系统和配备63x和/或100x物镜的ZEN 2010显微镜系统。
  18. 让显微镜和激光器在使用前预热15-20分钟。
  19. 用于染色的设置取决于核染色:
    1. DAPI(激光线:405)激发/发射350/470(nm);
    2. 碘化丙啶激发/发射535/617(nm);
    3. Hoechst 33342激发/发射350/461(nm);
    4. DRAQ5激发/发射647/681(nm)。
  20. 首先使用63x放大物镜找到核并捕获最少500个核。为了更好地解决后期桥梁,可以使用100x的目标
  21. 对于统计分析,a)有丝分裂(M),b)后期(A)和c)具有桥(AB)的后期分别计数细胞核(图1a和1b)。

    图1.具有后期桥的肿瘤细胞的荧光测定。。显示了有丝分裂(M)和具有后期桥(AB)的特定后期的细胞。比例尺=10μm。 b。通过在特定细胞周期阶段扩增这些细胞核可以鉴定明显的差异。显示了不同细胞周期阶段的细胞核的代表性图像。 A.间期,B. Prometaphase,C.中期,D.后期和E.后期与桥梁。比例尺=5μm。所有细胞用DAPI染色。标记了后桥的"桥"(白色箭头)。

数据分析

后期桥的频率计算为表现后期桥的细胞的比率和计数至少100个后期的后期细胞的总数。例如,我们区分计算i)所有阶段中的所有细胞核,ii)所有细胞在有丝分裂中,iii)后期细胞,和iv)具有桥的后期(还请参见图1b)。为了统计意义,计算学生的 t 测试值 注意:对于该方案,我们如先前所述(Aschacher等人,2016)用乱序siRNA或针对LINE-1 mRNA的有效siRNA处理肿瘤细胞系。有效的LINE-1 siRNA治疗在三天内引起端粒损耗,并导致后期桥(表1和图2)。

表1.后期桥梁的统计计算。给出了后期桥梁计数的代表表。至少应进行三次独立实验。



图2.后期桥的统计可视化的图示。分别给出计数核(包括间期和有丝分裂),有丝分裂,后期和具有后期桥的细胞。每个计数的数字在条的顶部给出。

食谱

  1. 生长介质
    500 ml McCoy's 5A培养基
    50 ml FBS,热灭活
  2. 3.7%甲醛溶液
    1 ml 37%甲醛
    9 ml 1x PBS
  3. 渗透解决方案
    1x含0.25%Triton X-10的PBS

致谢

该协议主要是从出版物Aschacher等人修改的 。 (2016年)。这项工作得到了维也纳医科大学在外科研究实验室的支持。

参考文献

  1. Aschacher,T.,Wolf,B.,Enzmann,F.,Kienzl,P.,Messner,B.,Sampl,S.,Svoboda,M.,Mechtcheriakova,D.,Holzmann,K.and Bergmann, 2016)。  LINE-1诱导 hTERT <并确保肿瘤细胞系中的端粒维持。致癌基因35(1):94-104。
  2. 这样,我们就可以得到这样的结果:一个简单的方法是使用一个简单的方法, "http://www.ncbi.nlm.nih.gov/pubmed/24379413"target ="_ blank"> TopBP1/Dpb11结合DNA后期桥,以防止基因组不稳定。细胞生物 204(1):45-59。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Aschacher, T. and Enzmann, F. (2016). Detection of Anaphase Bridge Formation by Immunofluorescence Microscopy in Mammalian Cells. Bio-protocol 6(20): e1977. DOI: 10.21769/BioProtoc.1977.
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