Flow Cytometry of Yeasts

David Lloyd1

1 University of Wales, Cardiff, United Kingdom
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.10
DOI:  10.1002/0471142956.cy1110s09
Online Posting Date:  May, 2001
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Abstract

This unit promotes increased interest in the use of flow cytometry in several new environments. The author provides protocols and descriptive detail on measurements of cell cycle, viability, respiratory activity, and β‐galactosidase activity. Many of these assays are described for other biological systems in CPC, but are now provided in detail for yeasts. Despite the increasing usefulness of digital imaging techniques, flow cytometry remains the method of choice for the resolution of population heterogeneities.

     
 
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Table of Contents

  • Basic Protocol 1: Monitoring the Cell Division Cycle of Yeasts
  • Basic Protocol 2: Determining the Viability of Yeast
  • Basic Protocol 3: Evaluating the Mitochondrial (Respiratory) Function of Yeast
  • Basic Protocol 4: Assaying β‐Galactosidase Activity In Vivo
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Monitoring the Cell Division Cycle of Yeasts

  Materials
  • Washed yeast suspension
  • 70% (v/v) ethanol
  • Tris⋅Mg2+ buffer (see recipe)
  • 0.5% (w/v) pepsin solution (see recipe)
  • 10 µg/ml RNase A solution (see recipe)
  • 50 µg/ml PI solution (see recipe)

Basic Protocol 2: Determining the Viability of Yeast

  Materials
  • Yeast suspension
  • Tris⋅Mg2+ buffer (see recipe)
  • 1 µg/ml DiBAC 4(3) solution (see recipe)

Basic Protocol 3: Evaluating the Mitochondrial (Respiratory) Function of Yeast

  Materials
  • Washed yeast suspension at 1–5 × 106 organisms/ml
  • 10 mM glucose
  • 1 µg/ml rhodamine 123 solution (see recipe)

Basic Protocol 4: Assaying β‐Galactosidase Activity In Vivo

  Materials
  • 2% (v/v) Triton X‐100
  • Substrate solution: 2 mg/ml resorufin β‐D‐galactopyranoside (Molecular Probes) in dimethyl sulfoxide
  • PBS ( appendix 2A), prechilled to 0°C
  • 2% (w/v) bovine serum albumin (BSA; Sigma) in PBS (store and use at 0°C)
  • Yeast suspension (106 cells/ml) washed twice in PBS after centrifugation from growth medium and kept at 4°C
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Figures

Videos

Literature Cited

Literature Cited
   Agar, D.W. and Bailey, J.E. 1982. Cell cycle operation during batch growth of fission yeast populations. Cytometry 3:123‐128.
   Alberghina, L. and Porro, D. 1993. Quantitative flow cytometry: Analysis of protein distributions in budding yeast. A mini review. Yeast 9:815‐823.
   Bell, P.J.L., Deere, D., Shen, J., Chapman, B., Bissinger, P.H., Attfield, P.V., and Veal, D.A. 1998. A flow cytometric method for rapid selection of novel yeast hybrids. Appl. Environ. Microbiol. 64:1669‐1672.
   Brailsford, M. and Gatley, S. 1993. Rapid analysis of microorganisms using flow cytometry. In Flow Cytometry in Microbiology (D. Lloyd, ed.) pp. 171‐180. Springer‐Verlag, London.
   Bruetschy, A., Laurent, M., and Jacquet, R. 1994. Use of flow cytometry in oenology to analyse yeasts. Lett. Appl. Microbiol. 18:343‐345.
   Carlson, C.R., Grallert, B., Bernander, R., Stokke, T., and Boye, E. 1997. Measurement of DNA content in fission yeast. Yeast 13:1329‐1335.
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   Costello, G., Rodgers, L., and Beach, D. 1986. Fission yeast enters the stationary phase G0 state from either mitotic G1 or G2. Curr. Genet. 11:119‐125.
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