Detection of Mitotic Cells

Gloria Juan1, Zbigniew Darzynkiewicz2

1 Memorial Sloan‐Kettering Cancer Center, New York, New York, 2 Brander Cancer Research Institute, New York Medical College, Valhalla, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.24
DOI:  10.1002/0471142956.cy0724s28
Online Posting Date:  May, 2004
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Abstract

In preparation for cell division, nuclear chromatin undergoes a vital rearrangement required for the organization of chromosomes and their allocation to daughter cells. This process is initiated during G2 phase with the most remarkable morphological manifestation being chromatin condensation. This unit provides protocols for identification and quantification of mitotic cells based on immunocytochemical detection of histone H3 phosphorylated on Ser 10 (H3‐P), the critical event occurring during the G2 to M transition (essential for chromatin condensation), using anti‐H3‐P, a commercially available antibody to which apoptotic cells are not reactive, concurrently with differential staining of cellular DNA. Additionally an adaptation of this method used to stain cells mounted on microscope slides for analysis by multiparameter laser scanning cytometry is also presented.

Keywords: cell cycle; chromatin; histone; multiparameter laser scanning cytometery; mitosis; mitotic index

     
 
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Table of Contents

  • Basic Protocol 1: Flow Cytometric Analysis of Histone H3 Using anti‐H3‐P Antibodies
  • Alternate Protocol 1: Laser‐Scanning Cytometric Analysis of Histone H3 Using Anti‐H3‐P Antibodies
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Flow Cytometric Analysis of Histone H3 Using anti‐H3‐P Antibodies

  Materials
  • Cells to be analyzed: growing in culture ( appendix 3B) or dissociated from solid tumor (unit 5.2; but do not fix dissociated cells)
  • Phosphate buffered saline (PBS; appendix 2A)
  • 1% (v/v) formaldehyde in PBS (see appendix 2A for PBS)
  • 80% ethanol, –20°C
  • 0.25% (v/v) Triton X‐100 in PBS, pH 7.4 (store at 4°C)
  • Rinsing buffer: 1% (w/v) bovine serum albumin (BSA) in PBS, pH 7.4 (store at 4°C)
  • Primary antibody to phosphorylated histone H3 (anti‐H3‐P MAb; Sigma)
  • Mouse IgG2a (isotypic control)
  • Secondary antibody: FITC‐conjugated goat anti‐mouse IgG
  • PI staining buffer: 5 µg/ml propidium iodide (PI) and 200 µg/ml DNase‐free RNase A in PBS, pH 7.4, freshly prepared
  • 15‐ml conical tubes, silanized or polypropylene
  • Flow cytometer equipped with 488‐nm argon laser and filters for collection of green (530 ± 20‐nm) and red (>620‐nm) fluorescence
  • Additional reagents and equipment for trypsinizing cells ( appendix 3B)

Alternate Protocol 1: Laser‐Scanning Cytometric Analysis of Histone H3 Using Anti‐H3‐P Antibodies

  • Tissue culture medium with serum
  • 70% ethanol
  • Paraffin or gelatin‐based sealer for coverslips
  • Shandon Cytospin cytocentrifuge and Cytospin chambers
  • Coplin jars
  • Humid chamber (vessel with lid, containing moistened paper towels)
  • Slides and coverslips
  • Laser scanning cytometer (LSC) equipped with 488‐nm argon laser and filters for collection of green (530±20‐nm) and red (>620‐nm) fluorescence
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Figures

Videos

Literature Cited

Literature Cited
   Ajiro, K. and Nishimoto, T. 1985. Specific site of histone H3 phosphorylation related to the maintenance of premature chromosome condensation: Evidence for catalytically induced interchange of the subunits. J. Biol. Chem. 260:15379‐15381.
   Balhorn, R., Jackson, V., Granner, D., and Chalkley, R. 1975. Phosphorylation of the lysine‐rich histones throughout the cell cycle. Biochemistry 14:2504‐2511.
   Clevenger, C.V., Epstein, A.L., and Bauer, K.D. 1987. Quantitative analysis of a nuclear antigen in interphase and mitotic cells. Cytometry 8:280‐286.
   Darzynkiewicz, Z., Traganos, F. and Melamed, M.R. 1980. New cell compartments identified by multiparameter flow cytometry. Cytometry 1:98‐108.
   Darzynkiewicz, Z., Gong, J. Juan, G., Ardelt, B., and Traganos, F. 1996. Cytometry of cyclin proteins. Cytometry 23:1‐13.
   Darzynkiewicz, Z., Juan, G., Li, X., Gorczyca, W., Murakami, T., and Traganos, F. 1997. Cytometry in cell necrobiology: Analysis of apoptosis and accidental cell death. Cytometry 27:1‐20.
   Di Vinci, A., Geido, E., Pfeffer, U., Vidali, G., and Giaretti, W. 1993. Quantitative analysis of mitotic and early‐G1 cells using monoclonal antibodies against AF‐2 protein. Cytometry 14:421‐427.
   Gong, J., Traganos, F., and Darzynkiewicz, Z. 1995. Discrimination of G2 and mitotic cells by flow cytometry based on different expression of cyclins A and B1. Exp. Cell Res. 220: 226‐231.
   Gorczyca, W., Melamed, M.R., and Darzynkiewicz, Z. 1996. Laser scanning cytometer (LSC) analysis of fraction of labeled mitoses. Cell Prolif. 29:539‐547.
   Gurley, L.R., Walters, R.A., and Tobey, R.A. 1975. Sequential phosphorylation of histone subfractions in the Chinese hamster cell cycle. J. Biol Chem. 250:3936‐3944.
   Gurley, L.R., Walters, R.A., Barham, S.S., and Daeven, L.L. 1978. Heterochromatin and histone phosphorylation. Exp. Cell Res. 11:373‐383.
   Juan, G., Pan, W., and Darzynkiewicz, Z. 1996. DNA segments sensitive to single strand specific nucleases are present in chromatin of mitotic cells. Exp. Cell Res. 227:197‐202.
   Juan, G., Li, X., and Darzynkiewicz, Z. 1997. Correlation between DNA replication and expression of cyclins A and B1 in individual MOLT‐4 cells. Cancer Res. 57:803‐807.
   Juan, G., Traganos, F., James, W.M., Ray, J.M., Roberge, M., Sauve, D.M., Anderson, H., and Darzynkiewicz, Z. 1998. Histone H3 phosphorylation and expression of cyclins A and B1 measured in individual cells during their progression through G2 and mitosis. Cytometry 32:1‐8.
   Juan, G., Traganos, F., and Darzynkiewicz, Z. 1999. Histone H3 phosphorylation in human monocytes and during HL‐60 cell differentiation. Exp. Cell Res. 246:212‐220.
   Landberg, G., Tan, E.M., and Ross, G. 1990. Flow cytometric multiparameter analysis of proliferating cell nuclear antigen/cyclin and Ki‐67 antigen: A new view of the cell cycle. Exp. Cell Res. 187:111‐118.
   Luther, E. and Kamentsky, L.A. 1996. Resolution of mitotic cells using laser scanning cytometry. Cytometry 23:272‐278.
   Nusse, M., Beisker, W., Hoffman, C., and Tarnok, A. 1990. Flow cytometric analysis of G1‐ and G2/M‐phase subpopulations in mammalian cell nuclei using side scatter and DNA content measurements. Cytometry 11:813‐821.
   Paulson, J.R. and Taylor, S.S. 1982. Phosphorylation of histone 1 and 3 and nonhistone high mobility group 14 by an endogenous kinase in HeLa metaphase chromosomes. J. Biol. Chem. 257:6064‐6072.
   Roti Roti, J.L., Higashikubo, R., Blair, O.C., and Uygur, N. 1982. Cell‐cycle position and nuclear protein content. Cytometry 3:91‐96.
   von Holde, K.E. 1989. Chromatin. Springer‐Verlag, New York.
   Wolffe, A. 1992. Chromatin: Structure and Function. Academic Press, San Diego.
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