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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

Cancer Biology > Proliferative signaling > Cell biology assays > Cell cycle
Author: Hui Zhu
Hui ZhuAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: huizhu@stanford.edu
Bio-protocol author page: a32
3/20/2012, 8842 views, 0 Q&A, How to cite
DOI: http://dx.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, FACS, MPM-2 antigens

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.


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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