Cell Cycle Analysis of Yeasts

Margarida Fortuna1, Maria João Sousa1, Manuela Côrte‐Real1, Cecília Leão1, Alexandre Salvador2, Filipe Sansonetty2

1 Universidade do Minho, Braga, Portugal, 2 IPATIMUP, Porto, Portugal
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.13
DOI:  10.1002/0471142956.cy1113s13
Online Posting Date:  May, 2001
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Staining protocols generally designed for the flow cytometric analysis of the cell cycle in mammalian cells are frequently not satisfactory for quantification of the various cell‐cycle phases in yeasts. High CVs limit the accuracy of DNA content measurement and estimates of populations in cell‐cycle compartments. This unit describes a staining procedure for yeasts using the sensitive nucleic acid stain SYBR Green I, which binds to double‐stranded DNA with high selectivity and which has a much higher fluorescence quantum yield upon binding than most other commonly used fluorophores. The properties of this dye combined with optimized sample processing provide high‐resolution DNA analysis, with half‐peak CVs around 3 to 4% and clear‐cut identification of the S phase.

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Table of Contents

  • Basic Protocol 1: Cell Cycle Analysis of Yeasts Using SYBR Green I
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Cell Cycle Analysis of Yeasts Using SYBR Green I

  • Yeast cell suspension
  • 70% (v/v) ethanol
  • 50 mM sodium citrate buffer, pH 7.5 (see recipe)
  • 1 mg/ml RNase A solution (see recipe)
  • 20 mg/ml proteinase K in water (Boehringer Mannheim; store in aliquots at 4°C)
  • SYBR Green I working solution (see recipe)
  • Triton X‐100
  • Two sets of fluorescent microspheres, with nominal sizes and green fluorescence, giving scatter and fluorescence signals noncoincident with those of the cells of interest and with CVs ≤2.0%
  • 50°C water bath
  • Ultrasonic processor with a probe to sonicate small volumes of liquid (e.g., Jencons model GE50)
  • Flow cytometer with 488‐nm excitation and a band‐pass filter centered at 525 nm
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 3A)
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Literature Cited

Literature Cited
   Brock, T.D. 1973. Biología de los microorganismos. Ediciones Omega, S.A., Barcelona, Spain.
  This study was supported by a research grant (PAMAF no. 6088). We are very grateful to Alberto Alvarez‐Barrientos (Centro de Citometria de Flujo y Microscopia Confocal, Facultad de Farmacia, Universidad Complutense de Madrid, Spain), who kindly did the cell sorting experiments to validate the interpretation of the results obtained with the two‐parameter histograms relating green fluorescence signal area to signal height (peak).
   Carlson, C.R., Grallert, B., Bernander, R., Stokke, T., and Boye, E. 1997. Measurement of nuclear DNA content in fission yeast by flow cytometry. Yeast 13:1329‐1335.
   Dien, B.S., Peterson, M.S., and Srienc, F. 1994. Cell‐cycle analysis of Saccharomyces cerevisiae. Methods Cell Biol. 42:457‐475.
   Dujon, B. 1981. The Molecular Biology of the Yeast Saccharomyces (J.N. Strattem, E.W. Jones, and J.R. Broach, eds.) pp.505‐625. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Haase, S.B. and Lew, D.J. 1997. Flow cytometric analysis of DNA content in budding yeast. Methods Enzymol. 283:322‐332.
   Haugland, R.P. (ed.) 1999. Handbook of Fluorescent Probes and Research Chemicals, 7th ed. Molecular Probes, Eugene, Ore.
   Liao, R.S., Rennie, R.P., and Talbot, J.A. 1999. Assessment of the effect of amphotericin B on the vitality of Candida albicans. Antimicrob. Agents Chemother. 43:1034‐1041.
   Marie, D., Partensky, F., Jacquet, D., and Vaulot, D. 1997. Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I. Appl. Environ. Microbiol. 63:186‐193.
   Marie, D., Brussaard, C.P.D., Thyrhaug, R., Bratbak, G., and Vaulot, D. 1999. Enumeration of marine viruses in culture and natural samples by flow cytometry. Appl. Environ. Microbiol. 65:45‐52.
   Nishimura, Y., Higashiyama, T., Suzuki, L., Misumi, O., and Kuroiwa, T. 1998. The biparental transmission of the mitochondrial genome in Chlamydomonas reinhardtii visualized in living cells. Eur. J. Cell Biol. 77:124‐133.
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