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Fluorescence Activated Cell Sorting (FACS) is a sensitive method to count mitotic cells. Cells are stained with an antibody that recognizes an antigen present only in mitotic cells, combined with propidium iodide (PI) to stain DNA. Two-dimensional FACS scanning allows the differential quantitation of G2 and mitotic cells. Several antibodies to different mitotic markers have been used in the community, including antibodies to MPM-2 antigens present in mitotic cells. MPM-2 recognizes a phosphorylated epitope (LTPLK or YWFSPL) 6, 7 in a distinct class of phosphoproteins including MAP2, HSP70, cdc25, and DNA topoisomerase IIα, most of which are phosphorylated at the onset of mitosis. The commercial availability and specificity of antibodies to histone H3 phosphorylated at threonine 11, which is present only in mitotic cells, has also been widely used to detect mitosis cells.

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[Bio101] Mitotic Index Determined by FACS Protocol
[Bio101] 流式细胞术检测有丝分裂

癌症生物学 > 增殖信号转导 > 细胞生物学试验 > 细胞周期
作者: Hui Zhu
Hui ZhuAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: huizhu@stanford.edu
Bio-protocol author page: a32
3/20/2012, 10121 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.196

[Abstract] Fluorescence Activated Cell Sorting (FACS) is a sensitive method to count mitotic cells. Cells are stained with an antibody that recognizes an antigen present only in mitotic cells, combined with propidium iodide (PI) to stain DNA. Two-dimensional FACS scanning allows the differential quantitation of G2 and mitotic cells. Several antibodies to different mitotic markers have been used in the community, including antibodies to MPM-2 antigens present in mitotic cells. MPM-2 recognizes a phosphorylated epitope (LTPLK or YWFSPL) 6, 7 in a distinct class of phosphoproteins including MAP2, HSP70, cdc25, and DNA topoisomerase IIα, most of which are phosphorylated at the onset of mitosis. The commercial availability and specificity of antibodies to histone H3 phosphorylated at threonine 11, which is present only in mitotic cells, has also been widely used to detect mitosis cells.
Keywords: Mitotic cells(有丝分裂细胞), FACS(流式细胞仪), MPM-2 antigens(MPM-2抗原)

[Abstract] 利用流式细胞仪是一个非常灵敏的方法来检测有丝分裂细胞数。细胞可以被一种抗体染色,这种抗体只识别一种只在有丝分裂期表达的蛋白,并且用碘化丙啶染色DNA。二维的FACS可以扫描不同数量的G2和有丝分裂期的细胞。针对不同有丝分裂marker的抗体可以使用。MPM-2 的抗体已经被用过。MPM-2 可以识别磷酸化位点 (LTPLK or YWFSPL) 6, 7 在不同家族的磷酸蛋白类包括 MAP2, HSP70, cdc25和DNA拓扑异构酶 IIα,它们大部分会在有丝分裂起始被磷酸化。组蛋白H3在有丝分裂期间在苏氨酸11位点上被磷酸化,商业化的抗组蛋白H3的抗体已经被广泛应用检测有丝分裂细胞。

Materials and Reagents

  1. Phosphate buffered saline (PBS)
  2. Ethanol
  3. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  4. BSA (Sigma-Aldrich, catalog number: A3803 )
  5. MPM-2 monoclonal antibody
  6. Anti-phospho-Histone H3 (Thr11) (EMD Millipore, catalog number: 06-570 )
  7. Anti-phospho-Ser/Thr-Pro, MPM-2 (EMD Millipore, catalog number: 05-368 )
  8. Alexa 488-conjugated goat anti-mouse immunoglobulin G antibody (Life Technologies, Molecular Probes®/Alexa Fluor® 488, catalog number: A-11008 or A-11034 )
  9. RNase A (Sigma-Aldrich, catalog number: R4642 )
  10. PI (Sigma-Aldrich, catalog number: P-4170 )

Equipment

  1. Centrifuges (Beckman Falcon, TLS-55 )
  2. Incubator
  3. FACS machine
  4. FACS tubes (BD Biosciences, Falcon®, catalog number: 352054 )

Procedure

Note: All spins are done at 2,000 rpm for 5 min.

  1. After collecting the cells (of your choice), wash them with 500 μl of PBS once, resuspend in 150 μl of PBS and then add 350 μl ethanol. Mix and store cells at 4 °C for at least 1 h.
  2. Spin and remove ethanol. Resuspend cells in 500 μl of PBS containing 0.25% Triton X-100 and incubate on ice for 15 min.
  3. After centrifugeation, the cell pellet was suspended in 100 μl of PBS containing 1% BSA and 0.25 μg of Histone H3 monoclonal antibody or 0.06 µg MPM-2 monoclonal antibody and incubated for 1 h at room temperature (RT).
    Note: After this step, cell pellets become loose even after centrifugation, therefore it is better to use a pipet to remove the solutions rather than using an aspirator.
  4. Spin and wash with 150 μl of PBS containing 1% BSA once.
  5. Resuspend cells in Alexa 488-conjugated goat anti-mouse immuneoglobulin G antibody diluted at a ratio of 1:300 in 100 μl of PBS containing 1% BSA and incubate at RT in the dark for 30 min.
  6. After centrifugation, resuspend cells in 500 μl of PBS containing 10 μg ml-1 RNase A and 20 μg/ml PI, transfer to FACS tubes and incubate at RT in the dark for 30 min.
  7. Take sample to FACS immediately or store at 4 °C until FACS analysis.

Acknowledgments

This protocol was developed in the laboratory of Dr. Guowei Fang (Department of Biology, Stanford University, Stanford, CA, USA). This work was supported by a Burroughs-Wellcome Career Award in Biomedical Research (G.F.) and by grants from National Institutes of Health (GM062852 to G.F.).

References

  1. Field, C. M., Wuhr, M., Anderson, G. A., Kueh, H. Y., Strickland, D. and Mitchison, T. J. (2011). Actin behavior in bulk cytoplasm is cell cycle regulated in early vertebrate embryos. J Cell Sci 124(Pt 12): 2086-2095.
  2. Preuss, U., Landsberg, G. and Scheidtmann, K. H. (2003). Novel mitosis-specific phosphorylation of histone H3 at Thr11 mediated by Dlk/ZIP kinase. Nucleic Acids Res 31(3): 878-885.
  3. Qian, J., Lesage, B., Beullens, M., Van Eynde, A. and Bollen, M. (2011). PP1/Repo-man dephosphorylates mitotic histone H3 at T3 and regulates chromosomal aurora B targeting. Curr Biol 21(9): 766-773.

材料和试剂

  1. 磷酸盐缓冲盐水(PBS)
  2. 乙醇
  3. Triton X-100(Sigma-Aldrich,目录号:T9284)
  4. BSA(Sigma-Aldrich,目录号:A3803)
  5. MPM-2单克隆抗体
  6. 抗磷酸组蛋白H3(Thr11)(EMD Millipore,目录号:06-570)
  7. 抗磷酸-Ser/Thr-Pro,MPM-2(EMD Millipore,目录号:05-368)
  8. 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 />
  9. RNA酶A(Sigma-Aldrich,目录号:R4642)
  10. PI(Sigma-Aldrich,目录号:P-4170)

设备

  1. 离心机(Beckman Falcon,TLS-55)
  2. 孵化器
  3. FACS机器
  4. FACS管(BD Biosciences,Falcon ,目录号:352054)

程序

注意:所有旋转都是在2,000 rpm下进行5分钟。

  1. 收集细胞(你选择的)后,用500μlPBS洗一次,重悬在150μlPBS,然后加入350μl乙醇。混合并储存细胞在4°C至少1小时
  2. 旋转和去除乙醇。重悬细胞在500μl含有0.25%Triton X-100的PBS中,并在冰上孵育15分钟
  3. 离心后,将细胞沉淀悬浮于100μl含有1%BSA和0.25μg组蛋白H3单克隆抗体或0.06μgMPM-2单克隆抗体的PBS中,并在室温(RT)温育1小时。
    注意:此步骤后,即使离心后细胞沉淀也会变得松动,因此最好使用移液管除去溶液,而不是使用吸气器。
  4. 旋转并用150μl含有1%BSA的PBS洗涤一次
  5. 重悬细胞在Alexa 488结合山羊抗小鼠免疫球蛋白G抗体稀释比例为1:300在100微升含有1%BSA的PBS中,在室温下在黑暗中孵育30分钟。
  6. 离心后,将细胞重悬在500μl含有10μg/ml RNA酶A和20μg/ml PI的PBS中,转移到FACS管中,并在室温下在黑暗中孵育30分钟。 />
  7. 立即取样到FACS或存储在4°C直到FACS分析

致谢

这个协议是在国防部的实验室(斯坦福大学生物系,斯坦福,加利福尼亚州,美国)开发的。 这项工作得到了生物医学研究(G.F.)的Burroughs-Wellcome职业奖和国立卫生研究院(GM062852至G.F.)的资助。

参考文献

  1. Field,C.M.,Wuhr,M.,Anderson,G.A.,Kueh,H.Y.,Strickland,D。和Mitchison,T.J。(2011)。 大块细胞质中的肌动蛋白行为是早期脊椎动物胚胎中的细胞周期调节。 J Cell Sci 124(Pt 12):2086-2095。
  2. Preuss,U.,Landsberg,G。和Scheidtmann,K.H。(2003)。 由Dlk/ZIP激酶介导的Thr11组蛋白H3的新型有丝分裂特异性磷酸化。 Nucleic Acids Res 31(3):878-885
  3. Qian,J.,Lesage,B.,Beullens,M.,Van Eynde,A.和Bollen,M。(2011)。 PP1/Repo-man在T3处去磷酸化有丝分裂组蛋白H3,并调节染色体极光B靶向。 Curr Biol 21(9):766-773
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How to cite this protocol: Zhu, H. (2012). Mitotic Index Determined by FACS Protocol. Bio-protocol Bio101: e196. DOI: 10.21769/BioProtoc.196; Full Text



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