Yeast Cell Cycle Analysis: Combining DNA Staining with Cell and Nuclear Morphology

Meredith E.K. Calvert1, Joanne Lannigan2

1 Temasek Life Sciences Laboratory, National University of Singapore, Singapore, 2 University of Virginia, Charlottesville, Virginia
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.32
DOI:  10.1002/0471142956.cy0932s52
Online Posting Date:  April, 2010
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Abstract

In studies of eukaryotic cell cycle regulation, the budding yeast Saccharoymyces cerevisiae offers many advantages as a model system. Due to its simple growth requirements and genetic tractability, this organism is a powerful tool for investigating the molecular regulation of cell cycle control. One earlier disadvantage to performing cell cycle analyses in yeast was that existing methods were restricted to either visual analysis or flow cytometry, both of which present limitations in the scope and accuracy of the data obtained. This unit demonstrates the combined use of DNA content measurements and bright‐field image analysis using multispectral imaging flow cytometry (MIFC) to provide a more precise quantitation of yeast cell cycle distribution. The advantage of this method is the ability to analyze large numbers of cells using multiple cell cycle indicators in a relatively short amount of time. Curr. Protoc. Cytom. 52:9.32.1‐9.32.16. © 2010 by John Wiley & Sons, Inc.

Keywords: budding yeast; cell cycle; MIFC; multispectral imaging flow cytometry; ImageStream; morphometrics; morphology; image analysis; DNA content

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Yeast Cells for Cell Cycle Analysis
  • Support Protocol 1: Measurement of DNA Content and Acquisition of Bright‐Field Images Using the Imagestream MIFC
  • Support Protocol 2: Image Processing and Analysis Using the Ideas Software
  • Support Protocol 3: Building the Cell Cycle Data Analysis Template in the Ideas Software
  • Support Protocol 4: Performing Cell Cycle Analysis Using the Ideas‐Created Template
  • Support Protocol 5: Morphological Analysis: Bud Length
  • Reagents And solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Yeast Cells for Cell Cycle Analysis

  Materials
  • Yeast cells and appropriate medium
  • 70% ethanol/30% sorbitol
  • 50 mM sodium citrate (diluted from 0.5 M stock and filter sterilized)
  • 50 mM sodium citrate containing 0.5 mg/ml RNase A, prepare fresh
  • Pepsin solution (see recipe), prepare fresh
  • 50 mM SYTOX Green stock solution in DMSO (excitation maxima: 504 nm, emission maxima: 532 nm; Invitrogen, cat. no. S7020)
  • 30°C shaker
  • Spectrophotometer
  • 0.5‐ and 1.5‐ml microcentrifuge tubes
  • Centrifuge
  • Sonicator

Support Protocol 1: Measurement of DNA Content and Acquisition of Bright‐Field Images Using the Imagestream MIFC

  Materials
  • Cells (see protocol 1)
  • ImageStream imaging flow cytometer system (Amnis) with IDEAS software
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Figures

Videos

Literature Cited

Literature Cited
   Calvert, M.E.K., Lannigan, J.A., and Pemberton, L.F. 2008. Optimization of yeast cell cycle analysis and morphological characterization by multispectral imaging flow cytometry. Cytom. Part A 73:825‐833.
   Fantes, P. and Beggs, J. (eds.) 2000. The Yeast Nucleus: Frontiers in Molecular Biology. Oxford University Press, New York.
   Fox, M.H. 1980. A model for the computer analysis of synchronous DNA distributions obtained by flow cytometry. Cytometry 1:71‐77.
   George, T.C., Basiji, D.A., Hall, B.E., Lynch, D.H., Ortyn, W.E., Perry, D.J., Seo, M.J., Zimmerman, C.A., and Morrissey, P.J. 2004. Distinguishing modes of cell death using the ImageStream multispectral imaging flow cytometer. Cytom. Part A 59:237‐245.
   George, T.C., Morrissey, P.J., Cui, C., Singh, S., and Fitzgerald‐Bocarsly, P. 2009. Measurement of cytoplasmic to nuclear translocation. Curr. Protoc. Cytom. 47:9.28.1‐9.28.15.
   Haase, S.B. 2003. Cell cycle analysis of budding yeast using SYTOX Green. Curr. Protoc. Cytom. 26:7.23.1‐7.23.4.
   Haase, S.B. and Lew, D.J. 1997. Flow cytometric analysis of DNA content in budding yeast. Methods Enzymol. 283:322‐332
   Haase, S.B. and Reed, S.I. 2002. Improved flow cytometric analysis of the budding yeast cell cycle. Cell Cycle 1:132‐136.
   Hutter, K.J. and Eipel, H.E. 1978. Flow cytometric determinations of cellular substances in algae, bacteria, moulds and yeasts. Antonie Van Leeuwenhoek 44:269‐282.
   Ortyn, W.E., Hall, B.E., George, T.C., Frost, K., Basiji, D.A., Perry, D.J., Zimmerman, C.A., Coder, D., and Morrissey, P.J. 2006. Sensitivity measurement and compensation in spectral imaging. Cytom. Part A 69:852‐862.
   Piatti, S., Lengauer, C., and Nasmyth, K. 1995. Cdc6 is an unstable protein whose de novo synthesis in G1 is important for the onset of S phase and for preventing a ‘reductional’ anaphase in the budding yeast Saccharomyces cerevisiae. EMBO J. 14:3788‐3799.
   Schwob, E. and Nasmyth, K. 1993. CLB5 and CLB6, a new pair of B cyclins involved in DNA replication in Saccharomyces cerevisiae. Genes Dev. 7:1160‐1175.
   Watson, J.V., Chambers, S.H., and Smith, P.J. 1987. A pragmatic approach to the analysis of DNA histograms with a definable G1 peak. Cytometry 8:1‐8.
   Zhang, H. and Siede, W. 2004. Analysis of the budding yeast Saccharomyces cerevisiae cell cycle by morphological criteria and flow cytometry. Methods Mol. Biol. 241:77‐91.
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