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Jan 2015

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Telomere Dysfunction Induced Foci (TIF) Analysis
端粒功能失调诱导病灶(TIF)分析   

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Abstract

Telomerase maintains telomeric DNA in eukaryotes during early developments, ~90% of cancer cells and some proliferative stem like cells. Telomeric repeats at the end of chromosomes are associated with the shelterin complex. This complex consists of TRF1, TRF2, Rap1, TIN2, TPP1, POT1 which protect DNA from being recognized as DNA double-stranded breaks. Critically short telomeres or impaired shelterin proteins can cause telomere dysfunction, which eventually induces DNA damage responses at the telomeres. DNA damage responses can be identified by antibodies to 53BP1, gammaH2AX, Rad17, ATM, and Mre11. DNA damage foci at uncapped telomeres are referred to as Telomere dysfunction-Induced Foci (TIFs) (de Lange, 2005; Takai et al., 2003).

The TIF assay is based on the co-localization detection of DNA damage by an antibody against DNA damage markers, such as gamma-H2AX, and telomeres using an antibody against one of the shelterin proteins such as TRF2 (Takai et al., 2003; de Lange, 2002; Karlseder et al., 1999). The method we describe here can be used in normal human and cancer cells.

Other commonly used methods-Telomere Restriction Fragment (TRF) Analysis (Mender and Shay, 2015b) and Telomere Repeat Amplification Protocol (TRAP) (Mender and Shay, 2015a)- in telomere biology can be found by clicking on the indicated links.

Keywords: Telomere dysfunction induced foci (端粒功能障碍致病灶), Telomeres (端粒酶), Telomerase (端粒酶), Telomeric DNA damage (端粒DNA损伤)

Materials and Reagents

  1. Lab-Tek II Chamber slides (Thomas Scientific, catalog number: 154461 )
  2. Cancer or normal human cells
  3. Triton X-100 (J.T. Baker Chemical Co., catalog number: 7-x198 )
     Octyl Phenol Ethoxylate (Avantor Performance Materials, J.T. Baker, catalog number: X198-07 )
  4. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
  5. Nonidet-P40 (Fluka BioChemika, catalog number: 74385 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 74385”.
  6. Fish gelatin blocking buffer, 10% (Amresco, catalog number: M319 )
  7. Bovine Serum Albumin (BSA), Fraction V (Gemini Bio-Products, catalog number: 700-106P )
  8. Anti-phospho-histone H2A.X (Ser139, mouse) Antibody (Merck Millipore Corporation, catalog number: 05-636 )
  9. Anti-TRF2 Antibody [EPR3517 (Ouellette et al., 2000)] (rabbit) (Abcam, catalog number: ab108997 )
  10. Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate (Invitrogen, catalog number: A-21206 )
    Note: Currently, it is “Thermo Fisher Scientific, NovexTM, catalog number: A-21206”.
  11. Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor® 568 conjugate (Invitrogen, catalog number: A-11004 )
    Note: Currently, it is “Thermo Fisher Scientific, NovexTM, catalog number: A-11004”.
  12. Vectashield® Mounting Medium with DAPI (Vector Laboratories, catalog number: H-1200 )
  13. Deltavision® Immersion Oil N=1.518 (GEHC, part no: 290291717 )
  14. Phosphate-buffered saline (PBS)
  15. MilliQ® water
  16. 10x PBS buffer-phosphate buffer saline (see Recipes)
  17. 1x PBST buffer (see Recipes)

Equipment

  1. Personal DeltaVision wide-field fluorescent microscope (GE Healthcare, model: PD11435 )
  2. Lamp (Xenon)
  3. Camera (Photometrics)
  4. 60x/1.42 N.A. (numerical aperture) objective (Olympus)

Software

  1. DeltaVision® SoftWoRx software (GE Healthcare)
  2. Autoquant® Software (Media Cybernetics)
  3. Imaris® Software (Bitplane Imaris)

Procedure

  1. Sample preparation
    1. Seed cells on chamber slides and incubate them until they are at the desired confluency (~70% confluency). However, It is important to seed them well separated to analyze individual cells. Avoid overlapping cells.
      Note: The amount described here is for a two well chamber slide. It can be adjusted to differing sizes of chamber slides. Shaking is not required for washing steps.
    2. Wash cells with 1 ml 1x PBS for 5 min.
    3. Fix cells with 4% paraformaldehyde (600 μl) for 10 min.
    4. Wash cells twice with 1 ml 1x PBS for 5 min.
    5. Permeabilize cells with 0.5% Nonidet-P40 (600 μl) for 10 min at room temperature.
    6. Wash cells three times with 1 ml 1x PBS for 5 min.
    7. Incubate cells with blocking solution (0.2% fish gelatin and 0.5% BSA) (600 μl) for 30 min at room temperature to reduce nonspecific binding.
      Note: Fish gelatin needs to be warmed up to room temperature before use.
    8. Incubate cells with primary antibodies (450 μl/well) diluted in blocking solution for either 1 h at room temperature or overnight at 4 °C in humidified chamber.
      Primary antibodies: gamma-H2AX: 1:1,000 dilution
      TRF2: 1:250 dilution
    9. Wash cells three times with 1x PBST for 5 min.
    10. Wash cells three times with 1x PBS for 5 min.
    11. Incubate cells with secondary antibodies (1:500 dilution for each) diluted in blocking solution for 40 min (450 μl/well) at room temperature.
    12. Wash cells six times with 1 ml 1x PBS for 5 min.
    13. Remove the chamber portion of slide.
    14. Counterstain with DAPI and seal the edges of the slides with nail polish.
      Notes:
      1. The bottle of mounting medium is supplied with a screw cap that has a drop dispenser pipet. One drop of mounting medium, approximately equal to 25 μl, is dispensed on the slide and then coverslipped.
      2. Coverslip should be carefully inverted to a drop of mounting medium on microscope slides to allow the mounting medium disperse over the slide.
    15. The slides can be viewed immediately after drying or stored at 4 °C up to a month/ -20 °C for a longer time.
    16. Image on a fluorescent microscope.

  2. Imaging with fluorescent microscope
    Images can be acquired using a Personal DeltaVision® wide-field fluorescent microscope with an Olympus® 60x/1.42 N.A objective and a Coolsnap® HQ2 camera with an image size of 1,024 x 1,024. For best resolution, set the bin value for the camera to be 1 x 1 resulting in a pixel size of 0.1077 μm using the 60x/1.42 N.A objective. The optical section spacing between each z-stack should be approximately 0.15 μm. At least three channels can be sequentially captured with the TRITC (excitation: 555/28 nm, emission: 617/73 nm), FITC (excitation: 490/20 nm, emission: 528/38 nm) and DAPI (excitation: 360/40 nm, emission: 457/50 nm) filterset. Exposure for each of those channels is selected such that it was well below the saturation limit of 4,095 for the maximum intensity value in that image.

  3. Image analysis
    Before analyzing images for co-localization of two different antibodies, the resolution of the z-stacks can be improved by de-convolving using a blind de-convolution algorithm in AutoquantX3® software. De-convolution is a computational method to process images, which are captured in a microscope by using series of optical sections (z-stacks), in a better contrast and resolution. During de-convolution, these series of optical sections are combined in three dimensions and improves the image quality by removing the blurry effect of microscope (Goodwin, 2014). Then, a background subtraction filter is applied in Imaris® software to improve the quality of the images before running the co-localization analysis. It is useful to maintain the same background subtraction settings for all images.

  4. Co-localization analysis
    Co-localization analysis is performed using a Bitplane Imaris. Background subtraction filter to improve the quality of the images and baseline subtraction filter to subtract the estimated baseline from the data. Coloc algorithm with channel 1 (gamma-H2AX) and channel 2 (TRF2) is selected in Imaris® (Costes et al., 2004). A Region of Interest (ROI) is selected using channel 3 (DAPI). The ROI is thresholded allowing the investigator to select the signals that are inside of the nucleus. Any signal represented by dashed lines is considered as background signal (Costes et al., 2004) (Figure 1). The algorithm allows calculation of the threshold values for channel 1 (gamma-H2AX) and channel 2 (TRF2) according to automatic co-localization analysis thus removing user bias. After automatic thresholding of both channels, a new coloc channel, channel 4, is built with ‘Build Coloc Channel’ button in the coloc analysis algorithm. After creating a surface for each nucleus (channel 3, DAPI), Spot function is used in Imaris® to generate new spots for channel 1 (gamma-H2AX), channel 2 (TRF2), and channel 4 (coloc). For creating new spots in the previous step, we generally select 0.2 microns as the diameter for the spots. The surface that is created earlier for Channel 3 (DAPI) is chosen as the ROI and a Split Spots into Surface Objects function, which is a matlab extension in Imaris, is applied to generate new spots. Using the above steps, the number of spots for each protein in every nucleus is calculated individually.


    Figure 1. This is an example figure to show 2D Histogram and the Define Region of Interest (ROI) function. The ROI using Channel 3 (DAPI) is thresholded such that the signal outside the nucleus represented by dashed lines is not used for the co-localization analysis calculations. This image does not reflect actual biology and is only for illustrative purposes.

    Steps for colocalization analysis in imaris software



Recipes

  1. 10x PBS buffer-phosphate buffer saline
    Powder for 5 L of 10x is ready to use for preparation of 5 L of concentrated 10x phosphate-buffered saline (PBS)
    Prepare 5 L milliQ® water and a PBS powder in it
    Then add large stir bar and place on stirrer until solids are dissolved
  2. 1x PBST buffer
    1x PBS with 0.1% Triton

Acknowledgments

Some of these protocols were adapted from previously published studies. We thank Zeliha Gunnur Dikmen for her help in acquisition of TRAP gel and Abhijit Bugde from the Live Cell Imaging Facility at UT Southwestern for his assistance with the imaging and analysis part of Telomere dysfunction Induced Foci (TIF) analysis.

References

  1. Costes, S. V., Daelemans, D., Cho, E. H., Dobbin, Z., Pavlakis, G. and Lockett, S. (2004). Automatic and quantitative measurement of protein-protein colocalization in live cells. Biophys J 86(6): 3993-4003.
  2. de Lange, T. (2002). Protection of mammalian telomeres. Oncogene 21(4): 532-540.
  3. de Lange, T. (2005). Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev 19(18): 2100-2110.
  4. Goodwin, P. C. (2014). Quantitative deconvolution microscopy. Methods Cell Biol 123: 177-192.
  5. Karlseder, J., Broccoli, D., Dai, Y., Hardy, S. and de Lange, T. (1999). p53- and ATM-dependent apoptosis induced by telomeres lacking TRF2. Science 283(5406): 1321-1325.
  6. Mender, I. and Shay, J. W. (2015a). Telomerase repeated amplification protocol (TRAP). Bio-protocol 5(22): e1657.
  7. Mender, I. and Shay, J. W. (2015b). Telomere restriction fragment (TRF) analysis. Bio-protocol 5(22): e1658.
  8. Takai, H., Smogorzewska, A. and de Lange, T. (2003). DNA damage foci at dysfunctional telomeres. Curr Biol 13(17): 1549-1556.

简介

端粒酶在早期发育期间在真核生物中维持端粒DNA,〜90%的癌细胞和一些增殖性干细胞。染色体末端的端粒重复与shelterin复合物相关。该复合物由TRF1,TRF2,Rap1,TIN2,TPP1,POT1组成,其保护DNA不被识别为DNA双链断裂。临界短的端粒或受损的遮蔽蛋白可引起端粒功能障碍,其最终在端粒诱导DNA损伤反应。 DNA损伤反应可以通过抗53BP1,gammaH2AX,Rad17,ATM和Mre11的抗体来鉴定。未封端的端粒的DNA损伤灶被称为端粒功能障碍诱导的Foci(TIF)(de Lange,2005; Takai等人,2003)。
TIF测定基于使用针对遮蔽蛋白如TRF2之一的抗体对抗DNA损伤标记物例如γ-H2AX和端粒的抗体的DNA损伤的共定位检测(Takai等人,2003; de Lange,2002; Karlseder等人,1999)。我们在这里描述的方法可以用于正常的人类和癌细胞。
其他常用的方法 - Telomere限制性片段(TRF)分析(Mender和Shay,2015b)和端粒重复扩增方案(TRAP) (Mender and Shay,2015a) - 通过点击指示的链接可以找到端粒生物学。

关键字:端粒功能障碍致病灶, 端粒酶, 端粒酶, 端粒DNA损伤

材料和试剂

  1. Lab-Tek II室幻灯片(Thomas Scientific,目录号:154461)
  2. 癌症或正常人类细胞
  3. Triton-X100(J.T.Baker Chemical Co.,目录号:7-x198)  辛基苯酚乙氧基化物(Avantor Performance Materials,J.T.Baker,目录号:X198-07)
  4. 多聚甲醛(PFA)(Sigma-Aldrich,目录号:P6148)
  5. Nonidet-P40(Fluka BioChemika,目录号:74385)
    注意:目前,它是"Sigma-Aldrich,目录号:74385"。
  6. 鱼明胶封闭缓冲液,10%(Amresco,目录号:M319)
  7. 牛血清白蛋白(BSA),组分V(Gemini Bio-Products,目录号:700-106P)
  8. 抗磷酸组蛋白H2A.X(Ser139,小鼠)抗体(Merck Millipore Corporation,目录号:05-636)
  9. 抗TRF2抗体[EPR3517(Ouellette等人,2000)](兔)(Abcam,目录号:ab108997)
  10. 驴抗兔IgG(H + L)第二抗体,Alexa Fluor 488缀合物(Invitrogen,目录号:A-21206)
    注意:目前,"赛默飞世尔科技,Novex TM ,目录号:A-21206"
  11. 山羊抗小鼠IgG(H + L)二抗,Alexa Fluor 568缀合物(Invitrogen,目录号:A-11004)
    注意:目前,"Thermo Fisher Scientific,Novex TM ,目录号:A-11004"
  12. 使用DAPI(Vector Laboratories,目录号:H-1200)的Vectashield 固定介质
  13. Deltavision 浸油N = 1.518(GEHC,件号:290291717)
  14. 磷酸盐缓冲盐水(PBS)
  15. MilliQ ?
  16. 10x PBS缓冲液 - 磷酸盐缓冲盐水(见配方)
  17. 1x PBST缓冲区(参见配方)

设备

  1. 个人DeltaVision宽场荧光显微镜(GE Healthcare,型号:PD11435)
  2. 灯(氙)
  3. 相机(Photometrics)
  4. 60x/1.42 N.A.(数值孔径)物镜(Olympus)

软件

  1. DeltaVision ? SoftWoRx软件(GE Healthcare)
  2. Autoquant ?软件(Media Cyber??netics)
  3. Imaris ?软件(位面板Imaris)

程序

  1. 样品准备
    1. 种子细胞在室幻灯片上,孵化他们,直到他们在 期望融合(?70%融合)。然而,种子很重要 它们很好地分离以分析个体细胞。避免重叠 细胞。
      注意:这里描述的量是一个两井室 滑动。它可以调整为不同尺寸的腔室滑块。摇晃 不需要清洗步骤。
    2. 用1ml 1×PBS洗涤细胞5分钟
    3. 用4%多聚甲醛(600μl)固定细胞10分钟
    4. 用1ml 1×PBS洗涤细胞两次,每次5分钟
    5. 在室温下用0.5%Nonidet-P40(600μl)透化细胞10分钟
    6. 用1ml 1×PBS洗涤细胞3次,每次5分钟
    7. 孵育细胞与封闭溶液(0.2%鱼明胶和0.5% BSA)(600μl)在室温下30分钟以减少非特异性 绑定 注意:鱼明胶需要在使用前温热至室温。
    8. 孵育细胞与一抗(450微升/孔)稀释 封闭溶液在室温下孵育1小时或在4℃过夜 ℃。
      一抗:γ-H2AX:1:1,000稀释
      TRF2:1:250稀释
    9. 用1x PBST洗涤细胞3次,每次5分钟
    10. 用1x PBS洗涤细胞3次,每次5分钟。
    11. 用二级抗体孵育细胞(每份1:500稀释) 在封闭溶液中稀释40分钟(450μl/孔) 温度
    12. 用1ml 1×PBS洗涤细胞6次,每次5分钟
    13. 取出载玻片的腔室部分。
    14. 用DAPI复染并用指甲油密封载玻片的边缘 注意:
      1. 安装介质瓶配有一个螺帽,有一个 ?滴分配器移液器。一滴安装介质,大约相等 至25μl,分配在载玻片上,然后盖上盖玻片。
      2. 盖玻片应小心地倒置到一滴安装介质上 显微镜载玻片以允许安装介质分散在载玻片上
    15. 干燥后可以立即观察载玻片,或者在4°C下长达一个月/-20°C更长时间保存。
    16. 在荧光显微镜上的图像。

  2. 用荧光显微镜成像
    可以使用具有Olympus 60x/1.42NA物镜的个人DeltaVision 宽视场荧光显微镜和Coolsnap sup> 2 相机,图像大小为1,024 x 1,024。为了获得最佳分辨率,请使用60x/1.42 N.A物镜将相机的像素值设置为1 x 1,从而使像素大小为0.1077μm。每个z堆叠之间的光学截面间隔应该大约为0.15μm。至少三个通道可以用TRITC(激发:555/28nm,发射:617/73nm),FITC(激发:490/20nm,发射:528/38nm)和DAPI(激发:360/40nm,发射:457/50nm)滤波器组。选择这些通道中的每一个的曝光,使得其远低于该图像中的最大强度值的4,095的饱和限度。

  3. 图像分析
    在分析用于两种不同抗体的共定位的图像之前,可以通过使用AutoquantX3软件中的盲去卷积算法去卷积来提高z-堆叠的分辨率。反卷积是一种处理图像的计算方法,其通过使用一系列光学截面(z-堆叠)在显微镜中捕获,以更好的对比度和分辨率。在去卷积过程中,这些系列的光学部分在三维上结合,通过消除显微镜的模糊效应来改善图像质量(Goodwin,2014)。然后,在Imaris 软件中应用背景减除滤波器,以在运行共定位分析之前提高图像的质量。对所有图像保持相同的背景减除设置是有用的。

  4. 共定位分析
    使用Bitplane Imaris执行协同定位分析。背景减除滤波器以提高图像的质量,并且基线减法滤波器从数据中减去估计的基线。在Imaris中选择具有通道1(γ-H 2 AX)和通道2(TRF2)的Coloc算法 2004)。使用通道3(DAPI)选择感兴趣区域(ROI)。 ROI是阈值,允许研究者选择在细胞核内部的信号。由虚线表示的任何信号被认为是背景信号(Costes等人,2004)(图1)。该算法允许根据自动共定位分析计算通道1(γ-H2AX)和通道2(TRF2)的阈值,从而消除用户偏差。在两个通道的自动阈值化之后,在coloc分析算法中使用"Build Coloc Channel"按钮构建新的coloc通道,通道4。在为每个核(通道3,DAPI)创建表面之后,在Imaris 中使用Spot函数来产生通道1(gamma-H2AX),通道2(TRF2)和通道4 (coloc)。为了在前一步骤中产生新斑点,我们通常选择0.2微米作为斑点的直径。先前为通道3(DAPI)创建的表面被选择为ROI,并且应用分裂点到表面对象函数(其是Imaris中的matlab扩展)来生成新点。使用上述步骤,单独计算每个核中每种蛋白质的斑点数

    图1.这是示出2D直方图和定义感兴趣区域(ROI)函数的示例图。使用通道3(DAPI)的ROI被阈值化,使得由虚线表示的核外部的信号线不用于共定位分析计算。此图片不反映实际生物学,仅用于说明目的
    在imaris软件中进行colocalization分析的步骤



食谱

  1. 10x PBS缓冲液 - 磷酸盐缓冲盐水
    5L 10x的粉末准备用于制备5L浓缩的10x磷酸盐缓冲盐水(PBS)
    准备5 L milliQ 水和PBS粉末在其中
    然后加入大量搅拌棒,并置于搅拌器上,直到固体溶解
  2. 1x PBST缓冲区
    1x含0.1%Triton的PBS

确认

其中一些协议改编自以前发表的研究。我们感谢Zeliha Gunnur Dikmen帮助从UT西南的活细胞成像设备获得TRAP凝胶和Abhijit Bugde,他对端粒功能障碍诱导Foci(TIF)分析的成像和分析部分的帮助。

参考文献

  1. Costes,S.V.,Daelemans,D.,Cho,E.H.,Dobbin,Z.,Pavlakis,G.and Lockett,S。(2004)。 自动定量测量活细胞中的蛋白质 - 蛋白质共定位。 J 86(6):3993-4003。
  2. de Lange,T。(2002)。 保护哺乳动物端粒。 癌基因 21(4) :532-540。
  3. de Lange,T。(2005)。 Shelterin:形成并保护人类端粒的蛋白质复合物。 Genes Dev 19(18):2100-2110
  4. Goodwin,P.C。(2014)。 Quantitative deconvolution microscopy。 Methods Cell Biol 123:177 -192。
  5. Karlseder,J.,Broccoli,D.,Dai,Y.,Hardy,S。和de Lange,T。(1999)。 缺乏TRF2的端粒诱导的p53和ATM依赖性凋亡。科学 283(5406):1321-1325。
  6. Mender,I.和Shay,J.W。(2015a)。 端粒酶重复扩增方案(TRAP) 生物协议 5(22 ):e1657。
  7. Mender,I.和Shay,J. W.(2015b)。 端粒限制性片段(TRF)分析 生物协议 5(22 ):e1658。
  8. Takai,H.,Smogorzewska,A.and de Lange,T。(2003)。 DNA损伤聚焦于功能障碍的端粒。 Curr Biol 13 (17):1549-1556。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mender, I. and Shay, J. W. (2015). Telomere Dysfunction Induced Foci (TIF) Analysis. Bio-protocol 5(22): e1656. DOI: 10.21769/BioProtoc.1656.
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