Bivariate Analysis of DNA Content and Expression of Cyclin Proteins

Gloria Juan1, Zbigniew Darzynkiewicz1

1 New York Medical College, Valhalla, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.9
DOI:  10.1002/0471142956.cy0709s04
Online Posting Date:  May, 2001
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Abstract

Cyclins are key components of the cell cycle machinery. This unit describes techniques associated with measurement of these transiently expressed molecules and focusses on the combination of cell cycle analysis and expression of proliferation‐associated proteins via immunocytochemically attached antibodies.

Keywords: flow cytometry; cyclins; DNA content; cell cycle; proliferation‐associated proteins Cyclins are key components of the cell cycle machinery

     
 
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Table of Contents

  • Basic Protocol 1: Bivariate Analysis of DNA Content and Cyclins
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Bivariate Analysis of DNA Content and Cyclins

  Materials
  • Cells to be analyzed
  • Phosphate‐buffered saline (PBS; appendix 2A), pH 7.4
  • Fixative: 80% ethanol or 100% methanol, −20°C
  • Permeabilization solution: 0.25% (v/v) Triton X‐100 in PBS, pH 7.4 (store at 4°C)
  • Rinsing buffer: 1% bovine serum albumin (BSA) in PBS, pH 7.4 (store at 4°C)
  • Cyclin antibodies: e.g., mouse monoclonal antibodies to cyclin B1 (clone GNS‐1), cyclin A (clone BF‐683), cyclin D1 (clone G124‐326), cyclin D3 (clone G107‐565), and cyclin E (clone HE12; all provided by PharMingen; cyclin D1 may also be obtained from Immunotech)
  • Isotypic control: mouse IgG1
  • 1 g/liter FITC‐conjugated goat anti–mouse IgG antibody
  • PI staining buffer: 5 µg/ml PI and 200 µg/ml DNase‐free RNase A ( appendix 2A) in PBS, pH 7.4, made fresh
  • Silanized or polypropylene 15‐ml conical tube
  • Flow cytometer equipped with 488‐nm argon laser or mercury arc lamp with blue (BG12) excitation filter (∼50% cutoff at 470 nm)
  • Additional reagents and equipment for trypsinizing cells ( appendix 3B) or dissociating cells from tissues (unit 5.2)
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Figures

Videos

Literature Cited

Literature Cited
   Bauer, K.D. and Jacobberger, J.W. 1994. Analysis of intracellular proteins. Methods Cell Biol. 41:352‐373.
   Cardon‐Cardo, C. 1995. Mutations of cell cycle regulators. Biological and clinical implications for human neoplasis. Am. J. Pathol. 147:545‐560.
   Darzynkiewicz, Z., Gong, J., Juan, G., Ardelt, B., and Traganos, F. 1996. Cytometry of cyclin proteins. Cytometry 25:1‐13.
   Draetta, F.‐G. 1994. Mammalian G1 cyclins. Curr. Opin. Cell Biol. 6:842‐846.
   Gong, J., Traganos, F., and Darzynkiewicz, Z. 1993. Simultaneous analysis of cell cycle kinetics at two different DNA ploidy levels based on DNA content and cyclin B measurements. Cancer Res. 53:5096‐5099.
   Gong, J., Ardelt, B., Traganos, F., and Darzynkiewicz, Z. 1994a. Unscheduled expression of cyclin B1 and cyclin E in several leukemic and solid tumor cell lines. Cancer Res. 54:4285‐4288.
   Gong, J., Li, X., Traganos, F., and Darzynkiewicz, Z. 1994b. Expression of G1 and G2 cyclins measured in individual cells by multiparameter flow cytometry: A new tool in the analysis of the cell cycle. Cell Prolif. 27:357‐371.
   Gong, J., Bhatia, U., Traganos, F., and Darzynkiewicz, Z. 1995a. Expression of cyclins A, D2, and D3 in individual normal mitogen stimulated lymphocytes and in MOLT‐4 leukemic cells analyzed by multiparameter flow cytometry. Leukemia 9:983‐899.
   Gong, J., Traganos, F., and Darzynkiewicz, Z. 1995b. Growth imbalance and altered expression of cyclins B1, A, E, and D3 in MOLT‐4 cells synchronized in the cell cycle by inhibitors of DNA replication. Cell Growth Differ. 6:1485‐1493.
   Hartwell, L.H. and Kastan, M.B. 1994. Cell cycle control and cancer. Science 266:1821‐1823.
   Juan, G., Gong, J., Traganos, F., and Darzynkiewicz, Z. 1996. Unscheduled expression of cyclins D1 and D3 in human tumor cell lines. Cell Prolif. 29:259‐266.
   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.
   Lukas, J., Bartkova, J., Welcker, M., Petersen, O.W., Peters, G., Strauss, M., and Bartek, J. 1995. Cyclin D2 is a moderately oscillating nucleoprotein required for G1 phase progression in specific cell types. Oncogene 10:2125‐2134.
   Morgan, D.O. 1995. Principles of CDK regulation. Nature 374:131‐134.
   Norbury, C. and Nurse, P. 1992. Animal cell cycles and their control. Annu. Rev. Biochem. 61:441‐470.
   Pines, J. and Hunter, T. 1991. Human cyclin A and cyclin B are differentially located in the cell and undergo cell cycle–dependent nuclear transport. J. Cell Biol. 115:1‐17.
   Sherr, C.J. 1994. G1 phase progression. Cycling on cue. Cell 79:551‐555.
   Sherwood, S.W., Rush, D.P., Kung, A.L., and Schimke, R.T. 1994. Cyclin B1 expression in HeLa cells studied by flow cytometry. Exp. Cell Res. 211:275‐281.
   Urbani, L., Sherwood, S.W., and Schimke, R.T. 1995. Dissociation of nuclear and cytoplasmic cell cycle progression by drugs employed in cell synchronization. Exp.Cell Res. 219:159‐168.
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