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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.

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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

癌症生物学 > 通用技术 > 细胞生物学试验 > 增殖分析
作者: Hui Zhu
Hui ZhuAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: huizhu@stanford.edu
Bio-protocol author page: a32
2/5/2012, 10993 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.197

[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)

[Abstract] 溴脱氧尿苷(BrdU) 是一种胸苷类似物并且可以掺入心合成的S期细胞的DNA。BrdU 特异性抗体可以用于检测掺入的BrdU,从而可以表明细胞复制DNA和检测在S期细胞的比例。用免疫细胞化学检测掺入DNA中的BrdU是一种很有力的研究普通或者癌症细胞动力学的工具。用BrdU在体内或者体外标记癌细胞和接下来用BrdU特性性抗体检测掺入的BrdU是一种精确的应用广泛的方法来定量DNA的合成程度。

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.

材料和试剂

 

1.       BrdU (Sigma Catalog No.B-5002)

2.       PBS

3.       甲醇(Fisher Scientific, BP 1105-4)

4.       BSA (Sigma Catalog No. A 3803)

5.       HCl

6.       单克隆 anti-BrdU (Sigma Catalog No. B 2531)

7.       Alexa 488-conjugated 羊抗鼠二抗(Molecular Probes, Cat. No A-11008 or A-11034)

8.       DAPI (Boeringer Manheim 236 276, make 1 mg/ml stock, 1000×)

9.       盖玻片 (12 mm, Fisher 12-545-82)

10.   Fluormount-GTM (Mounting solution, Southern Biotechnology Associates, 0100-01)

 

仪器

 

1.        离心机

2.        封口膜

 

步骤

 

1.        分离细胞到组织培养板上,含有盖玻片或者小室的载玻片。

注意: a) 我通常加一个盖玻片放进24孔板中的一个孔中,或者不超过两个盖玻片在6孔板中的一个孔中,或者不超过5个盖玻片到60 mm中。 b) 用移液器吹打或者振动那个确保细胞不是全部集中在孔的中间,确保在盖玻片或者组织培养盘中没有气泡。.

2.        细胞生长到70-100 %融合。

3.         30 μM BrdU 到细胞中,孵育30-60 分钟。

注意: BrdU是光敏型,所以必须在黑暗中加入。细胞加了BraU以后也可能光敏感因此也在黑暗中培养。加入BraU的时间和浓度根据细胞的类型和复制时间来确定,范围在15分钟到2个小时,浓度从10 μM100 μM ,例如我们处理鼠胚胎成纤维细胞为30 μM 处理60min

4.        将盖玻片或者分室的载玻片到另一个含有足够甲醇(-20 储存)的组织板中,固定十分钟或者几周。

5.        小心的将盖玻片或者分室的载玻片从板中移出,并且将细胞一侧到封口膜上,转移后迅速用100ul PBS洗涤,不要让细胞干了。.

6.         100 μl PBS-BT封闭 30 min

7.        100 μl 2 M HCl, 孵化30 min

8.        100 μl PBS-BT洗两次, 每次 5 min

9.        100 μl  anti-BrdU (PBS-BT1:100 稀释),孵化 30 min

10.    100 μl PBS-BT洗三次, 每次 5 min

11.    40 μl 二抗 (1:200 dilution in PBS-BT)孵育30 min

12.    100 μl PBS-BT洗三次, 每次 5 min

13.    细胞在40 μl DAPI (终浓度为1 μg/ml 1:1000 ,用 PBS稀释) 孵育2 min, PBS 洗一次.

14.    5-10 μl 封固溶液去清洁显微镜载玻片, 将染色的盖玻片细胞面从一边放到封固溶液下,使得封固液完整的盖住整个盖玻片的表面,并且避免产生气泡。

15.    让封固溶液干了,并且自己在室温下封固 30 min.

 

配制方法

 

1.        BrdU 储存液在 PBS pH 7.4  ddH2O,过滤灭菌 (MW = 307.1 20 mM = 0.006 g/ml)

2.        PBS-BT溶液: 10 ml 10×PBS, 3 g BSA (to 3 %), 1 ml 10 % Triton X-100 (to 0.1 %), 1 ml 5 % NaN3, dH2O 100 ml, 储存于 4.

 

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How to cite this protocol: Zhu, H. (2012). Immunofluorescence Assay for S Phase Entry Using BrdU Incorporation. Bio-protocol Bio101: e197. DOI: 10.21769/BioProtoc.197; Full Text



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