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[Bio101] Immunofluorescence Assay for S Phase Entry Using BrdU Incorporation
[Bio101] 利用BrdU免疫荧光染色检测有丝分裂合成期

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Abstract

Bromodeoxyuridine (BrdU) is thymidine analogue and can incorporated into the newly synthezised DNA of S-phase cells, the antibody specific for BrdU can then be used to detect the incorporated BrdU, thus indicating cells that were actively replicating their DNA and estimating for the percentage of cells in S-phase. The immunocytochemical detection of BrdU incorporated into DNA is a powerful tool to study the cytokinetics of normal and tumor cells. In vitro or in vivo labeling of tumor cells with BrdU and the subsequent detection of incorporated BrdU with specific anti-BrdU monoclonal antibodies is an accurate and comprehensive method to quantitate the degree of DNA-synthesis.

Keywords: Bromodeoxyuridine(BrdU)(溴脱氧尿嘧啶核苷(BrdU)), S-phase(S期), Immunofluorescence(免疫荧光)

Materials and Reagents

  1. BrdU (Sigma-Aldrich, catalog number: B5002 )
  2. Phosphate buffered saline (PBS)
  3. Methanol (Thermo Fisher Scientific, catalog number: BP1105-4 )
  4. BSA (Sigma-Aldrich, catalog number: A3803 )
  5. HCl
  6. NaN3
  7. Triton X-100
  8. Monoclonal anti-BrdU (Sigma-Aldrich, catalog number: B2531 )
  9. Alexa 488-conjugated goat anti-mouse immunoglobulin G antibody (Life Technologies, Molecular Probes®/Alexa Fluor® 488, catalog number: A-11008 or A-11034 )
  10. DAPI (make 1 mg ml-1 stock, 1,000x) (Boeringer Manheim 236 276 )
  11. Coverglasses (12 mm) (Thermo Fisher Scientific, catalog number: 12-545-82 )
  12. Fluormount-GTM Mounting solution (SouthernBiotech, catalog number: 0100-01 )
  13. PBS-BT solution (see Recipes)

Equipment

  1. Centrifuges (Beckman Falcon, model: TLS-55 )
  2. Parafilm
  3. 6-well plate
  4. 24-well plate

Procedure

  1. Split cells onto tissue culture dishes containing coverglasses or chambered slides.
    Notes:
    1. We usually put one coverglass in one well of the 24-well plate; or no more than 2 coverglasses in one well of the 6-well plate, or no more than 5 coverglasses in a 60 mm dish.
    2. Pipette up and down or shaking the dish to make sure cells are not concentrating in the center of the dish well. Make sure there are no air bubbles between the coverglasses and the tissue culture dish.
  2. Grow cells to 70-100% confluency.
  3. Add 30 μM BrdU to cells. Incubate for 30-60 min.
    Note: BrdU is light sensitive and should be added in the dark. Cells pulsed with BrdU may be photosensitive -- incubations should be in the dark as well. Time of pulse and BrdU concentration variable with cell type and doubling time. Ranges from 15 min to 2 h, and from 10 μM to 100 μM. For example: We treat mouse embryonic fibroblast cells at 30 µM for 60 min.
  4. Transfer the coverglasses or chambered slides into another tissue plate containing sufficient methanol (-20 °C stock), fix for 10 min or a couple of weeks.
  5. Carefully transfer the coverglasses or chambered slides from the plates and place cell side up onto secured Parafilm. Wash the coverglasses or chambered slides with 100 µl PBS immediately after transfer, never dry the cells.
  6. Block with 100 µl PBS-BT for 30 min.
  7. Incubate 100 µl 2 M HCl, 30 min.
  8. Wash 2x with 100 µl PBS-BT, each wash for 5 min
  9. Incubate with 100 µl mouse anti-BrdU (1:100 dilution in PBS-BT), 30 min.
  10. Wash 3x with 100 µl PBS-BT, each wash for 5 min.
  11. Incubate with 40 µl second antibody (1:200 dilution in PBS-BT), 30 min.
  12. Wash 3x with 100 µl PBS-BT, each wash for 5 min.
  13. Cells were incubated in 40 µl DAPI (1 µg/ml final concentration, 1:1,000 dilute in PBS) for 2 min, and then wash with PBS once.
  14. Add 5-10 µl mounting solution to a clean microscope slide for each coverglass, place stained coverglass cell side down onto mounting solution from one edge; allow mounting solution to cover the entire surface of the coverglass, avoiding air bubbles.
  15. Let the mounting solution dry and self-seal for 30 min at RT.

Recipes

  1. BrdU stock in PBS (pH 7.4) or ddH2O, filter sterile (MW = 307.1, 20 mM = 0.006 g/ml)
  2. PBS-BT solution
    10 ml 10x PBS
    3 g BSA (to 3%)
    1 ml 10 % Triton X-100 (to 0.1%)
    1 ml 5% NaN3
    ddH2O to 100 ml, store at 4 °C.

Acknowledgments

This work was supported by the California Institute of Regenerative Medicine, Grant RL1-00100.

References

  1. Zhu, H., Coppinger, J. A., Jang, C. Y., Yates, J. R., 3rd and Fang, G. (2008). FAM29A promotes microtubule amplification via recruitment of the NEDD1-gamma-tubulin complex to the mitotic spindle. J Cell Biol 183(5): 835-848.

简介

溴脱氧尿苷(BrdU)是胸苷类似物并且可以并入S期细胞的新合成的DNA中,然后可以使用对BrdU特异性的抗体来检测掺入的BrdU,从而指示活跃地复制其DNA的细胞, 的S期细胞。 掺入DNA的BrdU的免疫细胞化学检测是研究正常和肿瘤细胞的细胞因子的有力工具。 在体外或体内用BrdU标记肿瘤细胞,随后用特异性抗BrdU单克隆抗体检测掺入的BrdU是一种准确和全面的方法, DNA合成。

关键字:溴脱氧尿嘧啶核苷(BrdU), S期, 免疫荧光

材料和试剂

  1. BrdU(Sigma-Aldrich,目录号:B5002)
  2. 磷酸盐缓冲盐水(PBS)
  3. 甲醇(Thermo Fisher Scientific,目录号:BP1105-4)
  4. BSA(Sigma-Aldrich,目录号:A3803)
  5. HCl
  6. NaN 3
  7. Triton X-100
  8. 单克隆抗BrdU(Sigma-Aldrich,目录号:B2531)
  9. Alexa 488-缀合的山羊抗小鼠免疫球蛋白G抗体(Life Technologies,Molecular Probes/Alexa Fluor <488,目录号:A-11008或A-11034) br />
  10. DAPI(制备1mg ml -1 -1原料,1,000x)(Boeringer Manheim 236 276)
  11. 盖玻璃(12mm)(Thermo Fisher Scientific,目录号:12-545-82)
  12. Fluormount-G TM 固定溶液(SouthernBiotech,目录号:0100-01)
  13. PBS-BT溶液(见配方)

设备

  1. 离心机(Beckman Falcon,型号:TLS-55)
  2. parafilm
  3. 6孔板
  4. 24孔板

程序

  1. 将细胞分开到含有盖玻片或有空隙的载玻片的组织培养皿上 注意:
    1. 我们通常在24孔板的一个孔中放置一个盖玻片; 或在6孔板的一个孔中不超过2个盖玻片,或在60mm皿中不超过5个盖玻璃。
    2. 上下移动或摇动培养皿以确保细胞不会集中在培养皿中心。 确保盖玻片和组织培养皿之间没有气泡。
  2. 生长细胞至70-100%汇合
  3. 向细胞中加入30μMBrdU。 孵育30-60分钟。
    注意:BrdU是光敏感的,应该在黑暗中添加。 用BrdU脉冲的细胞可以是光敏的 - 孵育也应该在黑暗中。 脉冲时间和BrdU浓度随细胞类型和倍增时间变化。 范围为15分钟至2小时,以及10μM至100μM。 例如:我们以30μM处理小鼠胚胎成纤维细胞60分钟。
  4. 将盖玻片或有腔室的幻灯片转移到另一个含有足够甲醇(-20°C股票)的组织板,固定10分钟或几个星期。
  5. 小心地转移盖玻片或有腔室的幻灯片从板,并将细胞侧向上到固定的石蜡膜。 转移后立即用100μlPBS洗涤盖玻片或带腔玻片,切勿干燥细胞
  6. 用100μlPBS-BT封闭30分钟
  7. 孵育100μl2 M HCl,30分钟
  8. 用100μlPBS-BT洗涤2次,每次洗涤5分钟
  9. 与100μl小鼠抗BrdU(在PBS-BT中1:100稀释)孵育30分钟
  10. 用100μlPBS-BT洗涤3次,每次洗涤5分钟
  11. 与40μl第二抗体(在PBS-BT中1:200稀释)孵育30分钟
  12. 用100μlPBS-BT洗涤3次,每次洗涤5分钟
  13. 将细胞在40μlDAPI(1μg/ml终浓度,在PBS中1:1000稀释)中温育2分钟,然后用PBS洗涤一次。
  14. 添加5-10微升安装溶液到每个盖玻片的干净的显微镜载玻片,将染色的盖玻片细胞侧向下放置在来自一个边缘的安装溶液上; 允许安装溶液覆盖盖玻片的整个表面,避免气泡
  15. 让安装溶液干燥并在室温下自密封30分钟。

食谱

  1. 过滤灭菌(MW = 307.1,20mM = 0.006g/ml)的PBS(pH 7.4)或ddH 2 O 2中的BrdU储液。
  2. PBS-BT溶液
    10ml 10×PBS
    3 g BSA(至3%)
    1ml 10%Triton X-100(至0.1%) 1ml 5%NaN 3

致谢

这项工作由加利福尼亚再生医学研究所,批准RL1-00100支持。

参考文献

  1. Zhu,H.,Coppinger,J.A.,Jang,C.Y.,Yates,J.R.,3rdand Fang,G。(2008)。 FAM29A通过募集NEDD1-γ-微管蛋白复合物到有丝分裂纺锤体促进微管扩增。 a> J Cell Biol 183(5):835-848。
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引用:Zhu, H. (2012). Immunofluorescence Assay for S Phase Entry Using BrdU Incorporation. Bio-protocol Bio101: e197. DOI: 10.21769/BioProtoc.197;
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