Cell Cycle Analysis of Budding Yeast Using SYTOX Green

Steven B. Haase1

1 Duke University, Durham, North Carolina
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.23
DOI:  10.1002/0471142956.cy0723s26
Online Posting Date:  November, 2004
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Flow cytometric determination of DNA content is a standard tool for cell cycle analysis in the budding yeast, Saccharomyces cerevisiae. Using SYTOX Green instead of propidium iodide (PI) in a standard ethanol fixation protocol increases the accuracy and reproducibility of cell cycle analyses. SYTOX‐stained cells exhibit improved coefficients of variation (CVs), a better correlation between DNA content and fluorescence, and decreased sensitivity to variances in experimental conditions when compared with PI‐stained cells. The SYTOX Green protocol provides researchers with a more accurate and reliable alternative to standard PI‐based protocols for the analysis of the budding yeast cell cycle.

Keywords: cell cycle; SYTOX Green; propidium iodide; flow cytometry

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1:

  Materials
  • ∼1 × 107 yeast cells grown in suspension
  • 95% (v/v) ethanol
  • RNase solution (see recipe)
  • Protease solution (see recipe)
  • 50 mM Tris·Cl, pH 8.0 ( appendix 2A)
  • 1 µM SYTOX Green staining solution (see recipe)
  • 15‐ml screw‐cap centrifuge tubes
  • 1.5‐ml microcentrifuge tubes
  • 37°C incubator or water bath
  • 12 × 75–mm tubes suitable for flow cytometer
  • Sonicator
  • Flow cytometer with 488‐nm excitation and a band‐pass filter appropriate for collecting fluorescence emission at 523 nm
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Guthrie, C. and Fink, G.R. (eds.) 1991. Guide to yeast genetics and molecular biology. Methods Enzymol. Vol. 169.
   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. 1979. Microbial DNA determinations by flow cytometry. J. Gen. Microbiol. 113:369‐375.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library