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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, 9207 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.

试剂和材料

 

1.        PBS

2.        乙醇

3.        Triton X-100 (Sigma Catalog No. T9284)

4.        BSA (Sigma Catalog No. A 3803)

5.        Anti-phospho-Histone H3 (Thr11) (Millipore, Cat. No. 06-570)

6.        Anti-phospho-Ser/Thr-Pro, MPM-2 (Millipore, Cat. No. 05-368)

7.        Alexa 488-接合 羊抗鼠免疫球蛋白g抗体 (Molecular Probes, Cat. No A-11008 or A-11034)

8.        RNase A  (Sigma Catalog No. R4642)

9.        碘化丙 (Sigma Catalog No. P-4170)

 

仪器

 

1.        离心机

2.        流式细胞仪

3.        FACS (BD Falcon No. 352054)

 

步骤

 

注意:所有的离心均为2000 rpm5 min.

1.        收集细胞后用 500 ul PBS 洗一次,用150ul PBS重悬,然后加350ul乙醇。混合并放在4度至少一个小时。

2.        离心除去乙醇,用含有0.25% Triton X-100 500ul PBS重悬,并且冰上孵育15分钟。

3.        离心后,细胞重悬在100 μl含有1% BSA 0.25 μg组蛋白H3的单克隆抗体或者0.06ug MPM-2 单克隆抗体的PBS中。在室温下孵育1个小时。

注意: 这步后,细胞组分变得松动,因此最好用移液器去去除溶液而不是用真空吸。, cell

4.        离心后用150 μl含有 1% BSA PBS 洗一次。

5.        100ulPBS(含有1% BSA 1300稀释的Alexa 488-接合羊抗鼠免疫球蛋白g抗体重选细胞,在黑暗下室温孵育30min

6.        离心后,用含有10 μg/ml RNase A 20 μg/ml 碘化丙500ulPBS重悬细胞。转移到FACS管中并且在黑暗下室温孵育30min 

7.        直接进行FACS试验或者存于4 °C

 

参考文献

 

1.         Qian J., Lesage B., Beullens M., Van Eynde A., Bollen M. (2011). PP1/Repo-Man dephosphorylates mitotic histone H3 at T3 and regulates chromosomal aurora B targeting. Current Biology 21(9): 766-73. 

2.         Preuss U., Landsberg G., Scheidtmann K.H. (2003). Novel mitosis-specific phosphorylation of histone H3 at Thr11 mediated by Dlk/ZIP kinase. Nucleic Acids Res 31(3): 878-85. 

3.         Field C.M., Wuhr M., Anderson G.A., Kueh H.Y., Strickland D., Mitchison T.J. (2011). Actin behavior in bulk cytoplasm is cell cycle regulated in early vertebrate embryos. Journal of Cell Science 124(Pt 12): 2086-95. 

 

 

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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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