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Spindle Angle Measurements
螺旋轴角度测量   

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Abstract

Spindle angles measures derive from the measures of spindle poles positions that were taken from fixed and immunostained adherent cells. To determine spindle angles (α), z-stack images of metaphasic cells immunostained with anti γ-tubulin (spindle poles) and anti β-tubulin antibodies (mitotic spindle) were acquired. A very simple ImageJ software macro was developed to measure the spindle angle using spindle pole coordinates (see Figure 1).

Keywords: Spindle(主轴), Angle(角), Mitosisi(mitosisi)



Figure 1. Spindle angle measurement principle. Spindle poles coordinates are measured, then the spindle angle alpha is calculated.

Materials and Reagents

  1. Fixed cells
  2. Antibodies
    For example, for mitotic spindle poles, an antibody against γ-tubulin antibody (clone AK-15) (e.g. Sigma-Aldrich, catalog number: T3320 )
    For mitotic spindle using an antibody directed against α-or β-tubulin (clone TUB 2.1) (e.g. Sigma-Aldrich, catalog number: T4026 )
    Anti-rabbit coupled to Alexa Fluor® 555 (Life Technologies, Invitrogen™, catalog number: A21429 )
    Anti-mouse coupled to Alexa Fluor® 488 (Life Technologies, Invitrogen™, catalog number: A11029 )
    Note: Primary (AK-15 and TUB 2.1) and secondary (Anti-rabbit coupled to Alexa Fluor® 555 and anti-mouse coupled to Alexa Fluor® 488) antibodies were used after a 1,000 time dilution in 1x PBS/1% BSA.
  3. 10x PBS (Life Technologies, Invitrogen™, catalog number: AM9625 )
  4. 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, catalog number: D9542 )
  5. BSA (Sigma-Aldrich, catalog number: A4503 )
  6. 1x PBS (see Recipes)
  7. 1x PBS/1% BSA (see Recipes)

Equipment

  1. We use a Zeiss Axioimager Z1 with 63x Plan-Apochromat 1.4 oil lens and using an Axiocam Mrm camera with a Grid Projection Illumination (apotome). The system is driven by Axiovision software. Images can also be obtained from any confocal microscopes/widefield microscope + deconvolution.
  2. 12 mm round coverslips (#1.5)
  3. Glass slides
  4. Vectashield® Mounting Media (Vector Laboratories, catalog number: H-1000 ) or ProLong® Gold (Life Technologies, Invitrogen™, catalog number: P36934 )

Software

  1. Axiovision software
  2. Open source software ImageJ 1.47q (http://rsbweb.nih.gov/ij/index.html)
  3. Macro for spindle angle measurements

Procedure

  1. Stain cells for spindle poles and mitotic spindle using the above-mentioned antibodies and according to the following protocol
    1. Cells grown on 12 mm round coverslips (#1.5) are fixed for 5 minutes at -20 °C in MeOH.
    2. Rehydrated in 1x PBS for 5 minutes and saturated in 1x PBS/1% BSA for at least 30 minutes at room temperature.
    3. Incubated for 2 hours at room temperature with primary antibodies in 1x PBS/1% BSA.
    4. After 3 washes of few seconds in 1x PBS/1% BSA, cells are incubated for 1 hour at room temperature with secondary antibodies coupled to fluorophores and DAPI (to stain DNA, DAPI was used at 0.5 μg/ml) in 1x PBS/1% BSA.
    5. After 3 washes in 1x PBS/1% BSA, coverslips are mounted onto glass slides using either Vectashield® or ProLong® Gold.

  2. Imaging analysis
    1. Acquire Z stacks with a 63x/100x PLAN APO lens. Use Nyquist/Shannon criterion for Z step calculation (0.24 μm for this lens). Image quality must be good enough so that poles are clearly identified.
    2. Make sure the acquisition software calibrates the image (i.e. voxel size is included in the image Metadata). If not, the macro will request to calibrate the Image. XY pixel size can be derived from (physical camera pixel size*camera binning)/(Objective Magnification*tube lens magnification).
    3. Save the Macro text into a 3Dangle.txt file in the Macro subfolder in the ImageJ directory. Install the macro using Plugins>Macros>Install. Select the point selection tool (if multipoint selection tool is selected, right click to switch). Double click on the point tool icon to select the automeasure option. Alternatively, run the macro once (Plugins>Macros>3Dangle).
    4. Using the point selection tool set as indicated in step B3, click on the two spindle poles. Then run the macro (Plugins>Macros>3Dangle). The macro uses the first two lines of the result table to compute the angle. The calculated angle is indicated in the log window. Then, the result table is cleared.

  3. Macro
    run("Point Tool...", "mark=0 auto-measure label selection=yellow");
    run("Set Measurements...", " redirect=None decimal=3");
    IsCalibratedImage();
    x1=getResult("X", 0);
    y1=getResult("Y", 0);
    z1=getResult("Slice", 0);
    x2=getResult("X", 1);
    y2=getResult("Y", 1);
    z2=getResult("Slice", 1);
    xmag1=x1;
    ymag1=y1;
    zmag1=0;
    xmag2=(x1-x2);
    ymag2=(y1-y2);
    zmag2=(z1-z2);
    // scalar product
    product = (xmag1*xmag2) + (ymag1*ymag2) + (zmag1*zmag2);
    // magnitude horizontal vector 1 (points 1- to (0,0,z1)
    length1 = sqrt(xmag1 * xmag1 + ymag1*ymag1 + zmag1*zmag1);
    // magnitude vector 2 (points 1-2)
    length2 = sqrt(xmag2 * xmag2 + ymag2*ymag2 + zmag2*zmag2);
    degrees = acos(product/length1/length2);
    print("3d angle is " + degrees + " degrees");
    run("Select None");
    run("Clear Results");
    exit();
    }

    function IsCalibratedImage()
    {
    um="um";
    getVoxelSize(width, height, depth, unit);
    if(unit=="pixels" || unit=="microns" || unit=="micron")
                 {
                 if (width==0 || width==1)
                             {
                             Dialog.create("Image Calibration:");
                             Dialog.addMessage("Image has to be calibrated \nPlease enter the following parameters");
                             Dialog.addNumber("x , y pixel size: ", 0, 3, 5, um);
                             Dialog.addNumber("Z step: ", 0, 3, 5, um);
                             Dialog.show();
                             XYscale=Dialog.getNumber();
                             Zscale=Dialog.getNumber();
                             n=nSlices;
                             run("Properties...", "channels=1 slices=n frames=1 unit=um pixel_width="+XYscale+"
                             pixel_height="+XYscale+" voxel_depth="+Zscale+" frame=[0 sec] origin=0,0");
                             return;
                             }
                 }
    }

Recipes

  1. 1x PBS
    Made by diluting 10x PBS in MilliQ water
  2. 1x PBS/1% BSA
    Made by addition of 1% (weight/volume) into 1x PBS

Acknowledgments

This protocol is adapted from: Bompard et al. (2013). GB was supported by a grant from ‘Fondation pour la Recherche Médicale’ (Université Montpellier 2). This work was supported by a grant MEGAPAK to NM from the ANR (Agence Nationale pour la Recherche) GENOPAT.

References

  1. Bompard, G., Rabeharivelo, G., Cau, J., Abrieu, A., Delsert, C. and Morin, N. (2013). P21-activated kinase 4 (PAK4) is required for metaphase spindle positioning and anchoring. Oncogene 32(7): 910-919.

简介

主轴角度测量值取自固定和免疫染色的贴壁细胞的纺锤体极点位置的测量值。 为了确定主轴角度(α),获得用抗γ-微管蛋白(纺锤极)和抗β-微管蛋白抗体(有丝分裂纺锤体)免疫染色的间期细胞的z-堆叠图像。 开发了一个非常简单的ImageJ软件宏,用主轴极坐标测量主轴角度(见图1)。

关键字:主轴, 角, mitosisi



图1.主轴角度测量原理。 测量主轴极坐标,然后计算主轴角度alpha。

材料和试剂

  1. 固定电池
  2. 抗体
    例如,对于有丝分裂纺锤体极,使用针对γ-微管蛋白抗体的抗体(克隆AK-15)(例如Sigma-Aldrich,目录号:T3320)
    对于使用针对α-或β-微管蛋白的抗体(克隆TUB 2.1)(例如Sigma-Aldrich,目录号:T4026)的有丝分裂纺锤体,
    与Alexa Fluor555(Life Technologies,Invitrogen TM,目录号:A21429)偶联的抗兔
    抗小鼠偶联Alexa Fluor 488(Life Technologies,Invitrogen TM,目录号:A11029)
    注意:初级(AK-15和TUB 2.1)和次级(抗兔偶联Alexa Fluor ® 555和抗小鼠偶联Alexa Fluor ® )抗体在1x PBS/1%BSA中稀释1,000倍后使用。
  3. 10x PBS(Life Technologies,Invitrogen TM,目录号:AM9625)
  4. 4',6-二脒基-2-苯基吲哚二盐酸盐(DAPI)(Sigma-Aldrich,目录号:D9542)
  5. BSA(Sigma-Aldrich,目录号:A4503)
  6. 1x PBS(请参阅配方)
  7. 1x PBS/1%BSA(参见配方)

设备

  1. 我们使用具有63x Plan-Apochromat 1.4油透镜的Zeiss Axioimager Z1和使用带网格投影照明(apotome)的Axiocam Mrm相机。 该系统由Axiovision软件驱动。 图像也可以从任何共聚焦显微镜/广场显微镜+解卷积获得
  2. 12毫米圆形盖玻片(#1.5)
  3. 玻璃滑槽
  4. Vectashield支架介质(Vector Laboratories,目录号:H-1000)或ProLong Gold(Life Technologies,Invitrogen TM,目录号:P36934)

软件

  1. Axiovision软件
  2. 开源软件ImageJ 1.47q( http://rsbweb.nih.gov/ij/index.html
  3. 用于主轴角度测量的宏

程序

  1. 使用上述抗体并根据以下方案使用纺锤体极和有丝分裂纺锤体的染色细胞
    1. 在12mm圆形盖玻片(#1.5)上生长的细胞在-20℃下在MeOH中固定5分钟
    2. 在1×PBS中再水化5分钟,并在1×PBS/1%BSA中在室温下饱和至少30分钟。
    3. 在室温下与在1x PBS/1%BSA中的一抗孵育2小时。
    4. 在1×PBS/1%BSA中洗涤几秒钟后,将细胞与偶联于荧光团和DAPI的第二抗体(以染色DNA,DAPI以0.5μg/ml使用)在1×PBS/1中温育1小时 %BSA。
    5. 在1×PBS/1%BSA中洗涤3次后,使用Vectashield 或ProLong Gold将盖玻片安装到载玻片上。

  2. 成像分析
    1. 使用63x/100x PLAN APO镜头采集Z堆栈。使用Nyquist/Shannon准则进行Z步进计算(对于该透镜为0.24μm)。图像质量必须足够好,才能清楚地识别极点。
    2. 确保采集软件校准图像(即体素大小包含在图像元数据中)。如果没有,宏将请求校准图像。 XY像素大小可以从(物理相机像素大小*相机像素化)/(物镜放大倍率*镜头放大倍数)导出。
    3. 将Macro文本保存到ImageJ目录中的Macro子文件夹中的3Dangle.txt文件中。使用插件>宏>安装安装宏。选择点选择工具(如果选择多点选择工具,右键单击切换)。双击点工具图标以选择自动选项。或者,运行宏一次(插件>宏> 3Dangle)。
    4. 使用步骤B3中指示的点选择工具集,点击两个主轴极点。然后运行宏(插件>宏> 3Dangle)。宏使用结果表的前两行来计算角度。计算的角度在日志窗口中指示。然后,结果表被清除。


  3. 运行("点工具...","标记= 0自动测量标签选择=黄色");
    运行("设置测量...","重定向=无十进制= 3");
    IsCalibratedImage();
    x1 = getResult("X",0);
    y1 = getResult("Y",0);
    z1 = getResult("Slice",0);
    x2 = getResult("X",1);
    y2 = getResult("Y",1);
    z2 = getResult("Slice",1);
    xmag1 = x1;
    ymag1 = y1;
    zmag1 = 0;
    xmag2 =(x1-x2);
    ymag2 =(y1-y2);
    zmag2 =(z1-z2);
    //scalar product
    product =(xmag1 * xmag2)+(ymag1 * ymag2)+(zmag1 * zmag2);
    //幅度水平矢量1(点1-到(0,0,z1)
    length1 = sqrt(xmag1 * xmag1 + ymag1 * ymag1 + zmag1 * zmag1);
    //magnitude vector 2(points 1-2)
    length2 = sqrt(xmag2 * xmag2 + ymag2 * ymag2 + zmag2 * zmag2);
    degrees = acos(product/length1/length2);
    打印("3d角是"+度+"度");
    运行("选择无");
    运行("清除结果");
    exit();
    }

    function IsCalibratedImage()
    {
    um ="um";
    getVoxelSize(width,height,depth,unit);
    if(unit =="pixels"|| unit =="micron"|| unit =="micron")
                  {
                  if(width == 0 || width == 1)
                              {
                              Dialog.create("Image Calibration:");
                              Dialog.addMessage("图像必须校准\ n请输入以下参数");
                              Dialog.addNumber("x,y pixel size:",0,3,5,um);
                              Dialog.addNumber("Z step:",0,3,5,um);
                              Dialog.show();
                              XYscale = Dialog.getNumber();
                              Zscale = Dialog.getNumber();
                              n = n切片;
                              run("Properties ...","channels = 1 slices = n frames = 1 unit = um pixel_width ="+ XYscale +"
                              pixel_height ="+ XYscale +"voxel_depth ="+ Zscale +"frame = [0 sec] origin = 0,0");
                             返回;
                             }
                  }
    }

食谱

  1. 1x PBS
    通过稀释10x PBS在MilliQ水中制成
  2. 1×PBS/1%BSA
    通过向1×PBS中加入1%(重量/体积)制成

致谢

该协议改编自:Bompard等人(2013)。 GB得到"Fondation pour la RechercheMédicale"(蒙彼利埃大学2)的资助。 这项工作得到了一个授权MEGAPAK到NM从ANR(Agence Nationale pour la Recherche)GENOPAT支持。

参考文献

  1. Bompard,G.,Rabeharivelo,G.,Cau,J.,Abrieu,A.,Delsert,C.and Morin,N.(2013)。 P21激活激酶4(PAK4)是中期主轴定位和锚定所必需的。 Oncogene 32(7):910-919
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Cau, J., Morin, N. and Bompard, G. (2013). Spindle Angle Measurements. Bio-protocol 3(19): e925. DOI: 10.21769/BioProtoc.925.
  2. Mannen, T., Yamashita, S., Tomita, K., Goshima, N. and Hirose, T. (2016). The Sam68 nuclear body is composed of two RNase-sensitive substructures joined by the adaptor HNRNPL. J Cell Biol 214(1): 45-59.
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