搜索

Detection of the Cell Proliferation Zone in Leaves by Using EdU
采用 EdU测定叶片细胞增殖区   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Visualization of nuclei in S-phase cells in tissues is important for not only cell cycle research but also developmental research because morphogenesis is usually achieved by a combination of cell proliferation and cell expansion. Recently, DNA labeling with 5-ethynyl-2′-deoxyuridine (EdU), which is an analog of thymidine, has been used to visualize nuclei in S-phase cells to assess the activity of cell proliferation during development of plants. EdU is efficiently incorporated into newly synthesized DNA, and detection of EdU is based on the covalent reaction between EdU and Alexa Fluor® dye, which is one of useful fluorescent dyes; this allows us to use mild conditions for the assay without any DNA denaturation. This method could be easily applicable, and, indeed, has been used for various model and non-model plant species. Here, we have described a protocol developed for the detection of nuclei in S-phase cells in leaves.

Keywords: Cell division(细胞分裂), Cell proliferation(细胞增殖), 5-ethynyl-2′-deoxyuridine (EdU)(5-ethynyl-2′-脱氧尿苷(EDU)), Rorippa aquatica(蔊菜空心菜)

Materials and Reagents

  1. Plant tissues
  2. Click-iT® EdU Alexa Fluor® 488 Imaging Kit (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: c10337 )
  3. 90 ml acetone (WAKO, catalog number: 019-00353 )
  4. 1x phosphate-buffered saline (PBS) (e.g. WAKO, catalog number: 314-90185 )
  5. 0.5% Triton X-100 in 1x PBS
  6. Plastic tubes (PCR tube or 1.5-ml tube)
  7. Aluminum foil
  8. 90% acetone (see Recipes)
  9. Fixative formalin-acetic acid-alcohol (FAA) (see Recipes)
  10. 10 mM EdU stock solution (see Recipes)
  11. 10 µM EdU solution (see Recipes)
  12. Alexa Fluor® 488 azide solution (see Recipes)
  13. Click-iT® EdU reaction buffer (see Recipes)
  14. Click-iT® EdU buffer additive (see Recipes)
  15. Reaction cocktail (see Recipes)

Equipment

  1. Microscope (e.g. Nikon, model: ECLIPSE 80i )
  2. Shaker
  3. Vacuum pump
  4. Desiccator
  5. Razor
  6. Tweezer

Procedure

  1. For the experiment, select healthy plants and use seedlings, dissected leaves or leaf primordia, depending on the aim of the experiment. If you need to use older (mature) leaves, cut the leaves using a razor to obtain small-sized leaf discs (Figure 1A).
  2. Incubate the tissues in PCR tubes filled with 100-200 µl 10 µM EdU solution for 2 to 3 h [Figure 1B; normally 2 h. Kotogány et al. (2010) showed that labeling with 10 µM EdU for 2 h is sufficient for slowly growing cultured cells]. Avoid soaking the tissues or seedlings thoroughly in the solution to ensure that DNA replication and cell division proceed with less stresses, if your plant material is not aquatic plant (Figure 1B).
  3. After incubation, transfer the tissues gently to 1.5 ml tubes filled with iced 1 ml 90% acetone by tweezers and incubate on ice for 10 min.
  4. Transfer the tissues gently to other 1.5 ml tubes by tweezers, and wash the tissues 3 times with 1 ml 1x PBS by using a pipette.
  5. Remove the supernatant and fix the tissues with FAA for 2 to 3 h (the time for fixation depends on the plant materials or tissues). Decompression treatment using a pressure reducing pump for 15 min from the beginning or brief centrifugation are effective for the fixation.
  6. Transfer the tissues gently to other 1.5 ml tubes by tweezers, and wash the tissues twice with 1 ml 0.5% Triton X-100 in 1x PBS by shaking (100-150 rpm) for 5 min.
  7. After washing, prepare the reaction cocktail. (Do not prepare the cocktail before washing in order to avoid reducing the activity of the cocktail.)
  8. Remove the supernatant and add the reaction cocktail to the tubes (generally, 50-100 µl/tube) and mix well by using a pipette.
  9. Incubate the reaction cocktail for 30 min without any agitation and protect from light by wrapping the tubes with aluminum foil. To enhance the detection sensitivity, repeat step 8 with a fresh 50-100 µl/tube reaction cocktail.
  10. After incubation, transfer the tissues gently to other 1.5 ml tubes by tweezers and wash the tissues 3 times with 1 ml 1x PBS by shaking (100-150 rpm) for 20 min; protect from light by wrapping the tubes with aluminum foil.
  11. EdU signals can be observed using a fluorescence microscope (Figure 1C; for the detection of EdU with Alexa Fluor® 488 azide, use 458-488-nm excitation with a green emission filter). EdU signals can be observed a few weeks or even months after the detection reaction if the tissues are stored in 1x PBS in dark, at 4 °C.


    Figure 1. Plant material soaking in 10 µM EdU solution and visualization of nuclei in S-phase cells in the leaves of Rorippa aquatica (Brassicaceae). A. A leaf and leaf discs. B. A shoot apex subtending leaf primordia soaking in 10 µM EdU solution. C. Result of DNA labeling with EdU in leaves. This figure was taken under 20x objective. Bars = 1 cm in (A) and (B) and 100 µm in (C)

Representative data

For representative data, please see the papers of Ichihashi et al. (2014) and Nakayama et al. (2014).

Notes

  1. Basically, perform all the steps at room temperature.
  2. In all the steps, work gently to maintain the shape and structure of leaf primordia and leaves.
  3. Nuclei in S-phase cells can be easily quantified by counting all the signals in a defined area.
  4. This method is applicable for various plant tissues to detect nuclei in S-phase cells (e.g. shoot apical meristem).

Recipes

  1. 90% acetone (100 ml)
    90 ml acetone
    10 ml H2O
  2. FAA (100 ml)
    10 ml formaldehyde (37-40%)
    5 ml glacial acetic acid
    50 ml ethanol
    35 ml H2O
  3. 10 mM EdU stock solution
    To prepare 10 mM EdU stock solution, add 2 ml of DMSO (Component C) to Component A in the kit and mix well
    Stored at -20 °C
    When stored as directed, this stock solution will remain stable for up to 1 year
  4. 10 µM EdU solution
    To prepare 10 µM EdU solution, dilute 10 mM EdU stock solution to 10 µM by using H2O
  5. Alexa Fluor® 488 azide solution
    To prepare a working solution of Alexa Fluor® 488 azide, add 70 µl of DMSO to component B in the kit and mix well
    Stored at -20 °C
    When stored as directed, this solution will remain stable for up to 1 year
  6. Click-iT® EdU reaction buffer
    To prepare Click-iT® EdU reaction buffer, add 3.6 ml of H2O to 400 µl of Click-iT® EdU Reaction Buffer (Component D) in the kit and mix well
    Stored at 4 °C
    When stored as directed, this 1x buffer will remain stable for up to 6 months
  7. Click-iT® EdU buffer additive
    To prepare Click-iT® EdU buffer additive, add 1 ml of H2O to 200 mg of Click-iT® EdU Reaction Buffer additive (Component F) in the kit and mix well
    Stored at -20 °C
    When stored as directed, this buffer additive will remain stable for up to 1 year
  8. Reaction cocktail
    It is important to add the ingredients in the order listed in the table to ensure that the reaction proceeds at an optimal rate
    Use the reaction cocktail as soon as possible

    Table 1. Reaction cocktail
    Reaction components
    Volume
    Click-iT® EdU reaction buffer
    227.5 µl
    CuSO4 (Component E)
    10 µl
    Alexa Fluor® 488 azide solution
    0.6 µl
    Click-iT® EdU buffer additive
    12.5 µl
    Total Approx.
    250 µl

Acknowledgments

The protocol was modified from Kotogány et al. (2010). We thank Ms. Rumi Amano for preparing the figures. This research was partially supported by Grants-in-Aid from the Japan Society for the Promotion of Science (JSPS) (KAKENHI Grant Numbers 22870031, 24247007, 24770047 and 25113002) and The Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan to S. K. and BIO-NEXT project from Okazaki Institute for Integrative Bioscience to K. K. and H. T. and by a Research Fellowship from JSPS to H. N..

References

  1. Ichihashi, Y., Kawade, K. and Tsukaya, H. (2014). Leaf blade and leaf petiole of Arabidopsis thaliana. In: Noguchi, T., Kawano, S., Tsukaya, H., Matsunaga, S., Sakai, A., Karahara, I. and Hayashi, Y. (eds.) Atlas of plant cell structure. Springer, 194-195.
  2. Kotogany, E., Dudits, D., Horvath, G. V. and Ayaydin, F. (2010). A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine. Plant Methods 6(1): 5.
  3. Nakayama, H., Nakayama, N., Seiki, S., Kojima, M., Sakakibara, H., Sinha, N. and Kimura, S. (2014). Regulation of the KNOX-GA gene module induces heterophyllic alteration in North American lake cress. Plant Cell 26(12): 4733-4748.

简介

组织中S期细胞中核的可视化对于不仅细胞周期研究而且对发育研究是重要的,因为形态发生通常通过细胞增殖和细胞扩增的组合来实现。 最近,用5-乙炔基-2'-脱氧尿苷(EdU)(其是胸苷的类似物)的DNA标记已用于在S期细胞中显现细胞核以评估植物发育过程中细胞增殖的活性。 EdU有效地并入新合成的DNA中,并且EdU的检测基于EdU和作为有用的荧光染料之一的Alexa Fluor 染料之间的共价反应; 这允许我们使用温和条件进行测定而没有任何DNA变性。 该方法可以容易地应用,并且实际上已经用于各种模型和非模型植物物种。 在这里,我们已经描述了一个协议开发用于检测叶中S期细胞中的核。

关键字:细胞分裂, 细胞增殖, 5-ethynyl-2′-脱氧尿苷(EDU), 蔊菜空心菜

材料和试剂

  1. 植物组织
  2. (Thermo Fisher Scientific,Molecular Probes TM ,目录号:c10337)
  3. 90ml丙酮(WAKO,目录号:019-00353)
  4. 1×磷酸盐缓冲盐水(PBS)(例如WAKO,目录号:314-90185)
  5. 0.5%Triton X-100的1x PBS溶液
  6. 塑料管(PCR管或1.5 ml管)
  7. 铝箔
  8. 90%丙酮(见配方)
  9. 固定福尔马林 - 乙酸 - 酒精(FAA)(参见配方)
  10. 10 mM EdU储备溶液(见配方)
  11. 10μMEdU溶液(参见配方)
  12. Alexa Fluor ? 488叠氮化物溶液(参见配方)
  13. Click-iT ? EdU反应缓冲液(参见配方)
  14. Click-iT ? EdU缓冲添加剂(参见配方)
  15. 反应鸡尾酒(见配方)

设备

  1. 显微镜(如尼康,型号:ECLIPSE 80i)
  2. 振动器
  3. 真空泵
  4. 干燥器
  5. Razor
  6. 镊子

程序

  1. 对于实验,选择健康植物并使用幼苗,解剖叶或叶原基,取决于实验的目的。如果你需要使用较老的(成熟的)叶子,使用剃刀切割叶子以获得小型叶片(图1A)。
  2. 孵育组织在装有100-200μl10μMEdU溶液的PCR管中2-3小时[图1B;通常2小时。 Kotogányem et al。 (2010)显示用10μMEdU标记2小时对于缓慢生长的培养细胞是足够的]。避免将组织或幼苗彻底浸泡在溶液中,以确保如果植物材料不是水生植物,DNA复制和细胞分裂进行的应力较小(图1B)。
  3. 孵育后,通过镊子轻轻地转移组织到1.5毫升填充有1毫升90%丙酮的管中,并在冰上孵育10分钟。
  4. 通过镊子轻轻转移组织其他1.5毫升管,并用1毫升1×PBS,用移液器洗涤组织3次。
  5. 取出上清液并用FAA固定组织2至3小时(固定的时间取决于植物材料或组织)。使用减压泵从开始减压处理15分钟或短暂离心对于固定是有效的
  6. 通过镊子轻轻转移组织其他1.5毫升管,用1毫升0.5%Triton X-100在1×PBS中洗涤组织两次,通过摇动(100-150 rpm)5分钟。
  7. 洗涤后,制备反应混合物。 (不要在洗涤前准备鸡尾酒,以避免降低鸡尾酒的活性。)
  8. 取出上清液,加入反应混合物到管中(一般为50-100μl/管),用移液管充分混匀。
  9. 无需任何搅拌孵育反应混合物30分钟,通过用铝箔包裹管保护光。为了提高检测灵敏度,用新鲜的50-100μl/管反应混合物重复步骤8
  10. 孵育后,通过镊子轻轻转移组织其他1.5毫升管,并用1毫升1×PBS洗涤组织3次,通过摇动(100-150 rpm)20分钟;通过用铝箔包裹管来保护光。
  11. 可以使用荧光显微镜观察EdU信号(图1C;对于使用Alexa Fluor 488叠氮化物的EdU检测,使用具有绿色发射滤光片的458-488nm激发)。如果组织在4℃下在黑暗中储存在1×PBS中,则在检测反应后几周或甚至几个月可以观察到EdU信号。


    图1.植物材料浸泡在10μMEdU溶液中并显现水生生物Rorippa aquatica(Brassicaceae)叶中S期细胞中的核。 A.叶和叶光盘。 B.茎尖朝向在10μMEdU溶液中浸泡的叶原基。 C.叶中EdU的DNA标记结果。这个数字是在20倍目标下拍摄的。 (A)和(B)中的条= 1cm,(C)中为100μm

代表数据

有关代表性数据,请参阅Ichihashi等人(2014年)和Nakayama等人(2014年)的论文。

笔记

  1. 基本上,在室温下执行所有步骤。
  2. 在所有步骤中,轻轻地工作以保持叶原基和叶的形状和结构
  3. S期细胞中的核可以通过计数在限定区域中的所有信号容易地定量
  4. 该方法适用于各种植物组织以检测S期细胞(例如芽顶端分生组织)中的核。

食谱

  1. 90%丙酮(100ml) 90 ml丙酮
    10ml H 2 O x/v
  2. FAA(100ml)
    10ml甲醛(37-40%) 5ml冰乙酸
    50ml乙醇 35ml H 2 O 2 /
  3. 10mM EdU储液
    为了制备10mM EdU储备溶液,向试剂盒中的组分A中加入2ml DMSO(组分C),并充分混合
    储存于-20°C
    当按指示储存时,该储备溶液将保持稳定长达1年。
  4. 10μMEdU溶液
    为了制备10μMEdU溶液,使用H 2 O稀释10mM EdU储液至10μM。
  5. Alexa Fluor ? 488叠氮化物溶液
    为了制备Alexa Fluor 488叠氮化物的工作溶液,向试剂盒中的组分B中加入70μlDMSO,并混匀
    储存于-20°C
    当按指示储存时,此溶液将保持稳定最长1年。
  6. Click-iT ? EdU反应缓冲区
    为了制备Click-iT EdU反应缓冲液,将3.6ml H 2 O加到400μl的Click-iT EdU反应缓冲液组分D),并混匀。
    储存在4°C
    按指示存储时,此1x缓冲区将保持稳定长达6个月
  7. Click-iT ? EdU缓冲添加剂
    为了制备Click-iT EdU缓冲添加剂,将1ml H 2 O加入到200mg Click-iT EdU反应缓冲液添加剂(组分F),并混匀。
    储存于-20°C
    当按指示储存时,该缓冲添加剂将保持稳定长达1年。
  8. 反应液
    重要的是按表中列出的顺序添加成分,以确保反应以最佳速率进行。
    尽快使用反应鸡尾酒

    表1.反应鸡尾酒
    反应成分

    Click-iT ? EdU反应缓冲区
    227.5微升
    CuSO 4(成分E)
    10微升
    Alexa Fluor ? 488叠氮化物溶液
    0.6μl
    Click-iT ? EdU缓冲添加剂
    12.5μl
    总计约
    250微升

致谢

该方案从Kotogány等人修改。 (2010)。我们感谢Rumi Amano女士准备数字。这项研究得到日本科学促进会(JSPS)(KAKENHI Grant号22870031,24247007,24770047和25113002)的Grants-in-Aid和来自促进和互助公司的私人科学研究促进基金日本学校SK和BIO-NEXT项目从冈崎综合生物科学研究所KK和HT和研究奖学金从JSPS到HN。

参考文献

  1. Ichihashi,Y.,Kawade,K.and Tsukaya,H。(2014)。拟南芥叶片和叶柄。 在Noguchi,T.,Kawano,S.,Tsukaya,H.,Matsunaga,S.,Sakai,A.,Karahara,I。和Hayashi,Y。(eds。)Atlas of plant cell structure em>。 Springer,194-195。
  2. Kotogany,E.,Dudits,D.,Horvath,G.V。和Ayaydin,F。(2010)。 用于检测植物细胞和组织中S期细胞周期进展的快速而稳定的测定法,使用乙炔基脱氧尿苷。植物方法 6(1):5.
  3. Nakayama,H.,Nakayama,N.,Seiki,S.,Kojima,M.,Sakakibara,H.,Sinha,N.and Kimura,S。(2014)。 KNOX-GA基因模块的调控诱导北美湖水芹中的叶绿素改变。植物细胞 26(12):4733-4748。
  • 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: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Nakayama, H., Kawade, K., Tsukaya, H. and Kimura, S. (2015). Detection of the Cell Proliferation Zone in Leaves by Using EdU. Bio-protocol 5(18): e1600. DOI: 10.21769/BioProtoc.1600.
提问与回复

(提问前,请先登录)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。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。