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[Bio101] Cell Proliferation Assay by Flow Cytometry (BrdU and PI Staining)
[Bio101] 流式细胞术检测细胞增殖(BrdU和 PI 染色)   

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Abstract

Cell Proliferation assays include an important set of fluorescence-based tests that can monitor cell health and cell division by evaluating DNA synthesis through thymidine incorporation. Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) is a synthetic nucleoside that is an analogue of thymidine. BrdU is commonly used in the detection of proliferating cells in living tissues. BrdU can be incorporated into the newly synthesized DNA of replicating cells (during the S phase of the cell cycle), substituting for thymidine during DNA replication. Antibodies specific for BrdU can then be used to detect the incorporated chemical, thus indicating cells that were actively replicating their DNA. Binding of the antibody requires denaturation of the DNA, usually by exposing the cells to acid or heat. The incorporation of BrdU is normally analyzed in flow cytometry by labelling with a conjugate anti-BrdU antibody and DNA dyes Propidium Iodide (PI) to perform cell cycle analysis.

Keywords: BrdU(BrdU), Propidium Iodide(碘化丙啶), Cell cycle(细胞周期)

Materials and Reagents

  1. BrdU (Sigma-Aldrich, catalog number: B5002 )
  2. RNase A (Sigma-Aldrich, catalog number: R4642 )
  3. Tween 20 (Thermo Fisher Scientific, catalog number: BP337-500 )
  4. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  5. BSA (Sigma-Aldrich, catalog number: A3803 )
  6. Propidium iodide (PI) (Sigma-Aldrich, catalog number: P4170 )
  7. Monoclonal anti-BrdU (Sigma-Aldrich, catalog number: B2531 )
  8. Goat-anti-Mouse IgG (Whole molecule) FITC conjugate (Sigma-Aldrich, catalog number: F0257 )
  9. Sodium Tetraborate (Na2B4O7•10H2O) (Sigma-Aldrich, catalog number: B9876 )
  10. 0.05% Trypsin-EDTA (Life Technologies, Gibco®, catalog number: 25300-062 )
  11. Phosphate buffered saline (PBS)
  12. EtOH
  13. HCl
  14. Sodium tetraborate (see Recipes)
  15. PBS/ 1% BSA (see Recipes)
  16. PI stock solution (see Recipes)

Equipment

  1. Centrifuges (Beckman Falcon, TLS-55 )
  2. Fluorescence Activated Cell Sorting (FACS) machine
  3. FACS tubes (BD Biosciences, Falcon®, catalog number: 352054 )

Procedure

  1. Culture cells under optimum growing conditions (log growth phase at critical cell density). Add BrdU at a final concentration of 30 μM. 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.
  2. Remove BrdU media. Rinse once with PBS.
  3. Trypsinize and harvest cells.
  4. Permeabilize cells: Rsuspend pellet in 0.3 ml PBS, agitate gently, then add 0.7 ml ice-cold 100% EtOH slowly. Mix gently with a 1 ml glass transfer pipette. Store at 4 °C at least 1 h.
    Note: The cell concentration following permeabilization should be approximately 106 cells/sample. Samples can be stored in EtOH for up to 2 weeks at 4 °C.
  5. Pellet cells. Aspirate supernatant completely.
    Note: All pellet cells below at 4,000 rpm, 2 min.
  6. Add 0.5 ml 2 N HCl/0.5% Triton X-100 and incubate 30 min at room temperature (RT).
  7. Pellet cells and remove supernatant. Resuspend cells in 0.5 ml 0.1 M sodium tetraborate for 2 min. Pellet cells.
  8. Wash cells once with 150 µl PBS/ 1% BSA.
  9. Resuspend cells in 50 μl 0.5% Tween 20/1% BSA/PBS. Add 10-20 μl (1 μg/106 cells) α-BrdU (mAb) and incubate for 1 h at RT.
  10. Pellet cells and wash cells once with 150 µl PBS/ 1% BSA.
  11. Pellet cells and resuspend in 50 μl 0.5% Tween 20/1% BSA/PBS. Add 5 μl (1 μg/106 cells) Goat anti-Mouse IgG-FITC. Incubate 30 min at RT.
  12. Pellet cells and transfer to FACS tubes.
  13. Resuspend pellet in 0.5 ml PBS containing 10 μg/ml RNase A and 20 μg/ml PI stock solution.
  14. Leave samples at RT for 0.5 h in the dark.
  15. FACS immediately or store at 4 °C.
    Notes: Flow cytometry setup controls needed:
    1. Cells stained with PI alone.
    2. Cells with no BrdU pulse, and only the antibodies added.

Recipes

  1. 0.1 M sodium tetraborate (pH 8.5)
    2 N HCl
    0.5% Trition X-100 (0.05 ml)
    1.67 ml HCl in 8.28 ml ddH2O.
  2. PBS/ 1% BSA
    0.1 mg BSA in 10 ml PBS.
  3. 0.5% tween 20/1% BSA/PBS
    0.05 ml Tween 20
    0.1 mg BSA in 9.5 ml PBS.
  4. 1 mg/ml PI stock solution
    10 mg PI
    10 ml ddH2O
    5. BrdU stock in PBS pH 7.4 or ddH2O, filter sterile (MW = 307.1, 20 mM = 0.006 g/ml)

Acknowledgments

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

References

  1. Hoy, C. A., Seamer, L. C. and Schimke, R. T. (1989). Thermal denaturation of DNA for immunochemical staining of incorporated bromodeoxyuridine (BrdUrd): critical factors that affect the amount of fluorescence and the shape of BrdUrd/DNA histogram. Cytometry 10(6): 718-725.

简介

细胞增殖测定包括一组重要的基于荧光的测试,可通过胸苷掺入评估DNA合成来监测细胞健康和细胞分裂。 溴脱氧尿苷(5-溴-2'-脱氧尿苷,BrdU)是是胸苷类似物的合成核苷。 BrdU通常用于检测活组织中的增殖细胞。 BrdU可以掺入到复制细胞的新合成的DNA中(在细胞周期的S期期间),在DNA复制期间替代胸苷。 然后可以使用BrdU特异性的抗体来检测掺入的化学物质,从而指示活跃地复制其DNA的细胞。 抗体的结合需要DNA变性,通常通过将细胞暴露于酸或热。 通常通过用缀合物抗BrdU抗体和DNA染料碘化丙啶(PI)标记以进行细胞周期分析,在流式细胞术中分析BrdU的掺入。

关键字:BrdU, 碘化丙啶, 细胞周期

材料和试剂

  1. BrdU(Sigma-Aldrich,目录号:B5002)
  2. RNA酶A(Sigma-Aldrich,目录号:R4642)
  3. 吐温20(Thermo Fisher Scientific,目录号:BP337-500)
  4. Triton X-100(Sigma-Aldrich,目录号:T9284)
  5. BSA(Sigma-Aldrich,目录号:A3803)
  6. 碘化丙啶(PI)(Sigma-Aldrich,目录号:P4170)
  7. 单克隆抗BrdU(Sigma-Aldrich,目录号:B2531)
  8. 山羊抗小鼠IgG(全分子)FITC缀合物(Sigma-Aldrich,目录号:F0257)
  9. 四硼酸钠(Na 2 B 4 O 7 O 10·10H 2 O)(Sigma-Aldrich,目录号: B9876)
  10. 0.05%胰蛋白酶-EDTA(Life Technologies,Gibco ,目录号:25300-062)
  11. 磷酸盐缓冲盐水(PBS)
  12. EtOH
  13. HCl
  14. 四硼酸钠(参见配方)
  15. PBS/1%BSA(参见配方)
  16. PI储备溶液(见配方)

设备

  1. 离心机(Beckman Falcon,TLS-55)
  2. 荧光活化细胞分选(FACS)机器
  3. FACS管(BD Biosciences,Falcon ,目录号:352054)

程序

  1. 在最佳生长条件下培养细胞(在临界细胞密度处的对数生长期)。 加入终浓度为30μM的BrdU。 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。
  2. 删除BrdU介质。 用PBS冲洗一次。
  3. 胰蛋白酶消化和收获细胞
  4. 渗透细胞:在0.3ml PBS中悬浮沉淀,轻轻搅拌,然后缓慢加入0.7ml冰冷的100%EtOH。 用1 ml玻璃移液管轻轻混匀。 在4℃下储存至少1小时。
    注意:透化后的细胞浓度应为约10 6个细胞/样品。 样品可以在4°C下储存在EtOH中达2周。
  5. 粒细胞。 完全吸出上清液 注意:所有沉淀细胞在4,000 rpm,2分钟。
  6. 加入0.5ml 2N HCl/0.5%Triton X-100,在室温(RT)下孵育30分钟
  7. 沉淀细胞并除去上清液。 重悬细胞在0.5ml 0.1M四硼酸钠中2分钟。 颗粒细胞。
  8. 用150μlPBS/1%BSA洗涤细胞一次
  9. 重悬细胞在50μl0.5%吐温20/1%BSA/PBS。 加入10-20μl(1μg/10 6个细胞)α-BrdU(mAb),并在室温下孵育1小时。
  10. 沉淀细胞并用150μlPBS/1%BSA洗涤细胞一次
  11. 沉淀细胞并重悬于50μl0.5%Tween 20/1%BSA/PBS中。 加入5μl(1μg/10 6个细胞)山羊抗小鼠IgG-FITC。 在室温下孵育30分钟。
  12. 将细胞转移到FACS管中
  13. 在含有10μg/ml RNA酶A和20μg/ml PI原液的0.5ml PBS中重悬沉淀。
  14. 将样品在室温下在黑暗中保持0.5小时
  15. FACS立即或在4°C下存储 注意:需要流式细胞仪设置控制:
    1. 单独用PI染色的细胞。
    2. 没有BrdU脉冲,且只有抗体添加的细胞。

食谱

  1. 0.1M四硼酸钠(pH8.5) 2 N HCl
    0.5%Trition X-100(0.05ml) 1.67ml HCl在8.28ml ddH 2 O中
  2. PBS/1%BSA
    0.1mg BSA的10ml PBS溶液
  3. 0.5%吐温20/1%BSA/PBS 0.05ml Tween 20 0.1mg BSA在9.5ml PBS中
  4. 1 mg/ml PI储液
    10 mg PI
    10ml ddH 2 O 2 / 5.过滤灭菌(MW = 307.1,20mM = 0.006g/ml)的在pH 7.4或ddH 2 O中的PBS中的BrdU储液。

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. Hoy,C.A.,Seamer,L.C。和Schimke,R.T。(1989)。 DNA的热变性用于掺入溴脱氧尿苷(BrdUrd)的免疫化学染色:影响 荧光和BrdUrd/DNA直方图的形状。 Cytometry 10(6):718-725。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhu, H. (2012). Cell Proliferation Assay by Flow Cytometry (BrdU and PI Staining). Bio-protocol Bio101: e198. DOI: 10.21769/BioProtoc.198;
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Hello,

I hope you are well. My name Nawroz and I am PhD student at Keele University. I am wondering if I can use the brdu for CLC cell line and could you please send me more information about this method for the cell line because I want to use it to determined cell proliferation.
I appreciate your help.
Kindest regards
Nawroz O. Kareem
10/19/2012 5:27:19 AM Reply
Wait, I cannot fathom it being so straigthofrward.
9/2/2012 5:16:16 PM Reply