Assaying Cell Cycle Status Using Flow Cytometry

Kang Ho Kim1, Joel M. Sederstrom2

1 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, 2 Cytometry and Cell Sorting Core, Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 28.6
DOI:  10.1002/0471142727.mb2806s111
Online Posting Date:  July, 2015
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Abstract

In this unit, two protocols are described for analyzing cell cycle status using flow cytometry. The first is based on the simultaneous analysis of proliferation‐specific marker (Ki‐67) and cellular DNA content, which discriminate resting/quiescent cell populations (G0 cell) and quantify cell cycle distribution (G1, S, or G2/M), respectively. The second is based on differential staining of DNA and RNA through co‐staining of Hoechst 33342 and Pyronin Y, which is also useful to identify G0 cells from G1 cells. Along with these methods for analyzing cell cycle status, two additional methods for cell proliferation assays with recent updates of newly developed fluorophores, which allow multiplex analysis of cell cycle status, cell proliferation, and a gene of interest using flow cytometry, are outlined. © 2015 by John Wiley & Sons, Inc.

Keywords: cell cycle; flow cytometry; Ki‐67; propidium iodide; Pyronin Y; Hoechst 33342

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Flow Cytometric Analysis of Ki‐67 and DNA Content for Analyzing Cell Cycle Status
  • Alternate Protocol 1: Simultaneous Staining of Cell Surface Antigens with Ki‐67 and PI
  • Basic Protocol 2: Pyronin Y and Hoechst 33342 Staining for Analyzing Cell Cycle Status
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Flow Cytometric Analysis of Ki‐67 and DNA Content for Analyzing Cell Cycle Status

  Materials
  • Cells
  • 1× phosphate‐buffered saline (PBS)
  • 70% ethanol (−20°C)
  • FACS buffer (see recipe)
  • FITC‐conjugated anti‐Ki‐67 antibody
  • PI staining solution (see recipe)
  • Centrifuge
  • Flow cytometer equipped with 488‐nm blue laser and appropriate filter sets detecting FITC and PI fluorescence

Alternate Protocol 1: Simultaneous Staining of Cell Surface Antigens with Ki‐67 and PI

  Additional Material (also see protocol 1)
  • Fluorophore‐conjugated antibody against surface antigen
  • Fixation solution (4% paraformaldehyde)
  • Permeabilization solution (see recipe)
  • Saponin wash buffer (see recipe)
  • PI/saponin staining solution (see recipe)
NOTE: Other fixatives and permeabilization buffers are commercially available, e.g., FIX & PERM cell fixation and cell permeabiliazation kit (Life Technologies, cat. no. GAS003/GAS004); Cytofix/Cytoperm fixation/permeabiliazation solution kit (BD Biosciences, cat. no. 554714); etc. For detailed procedures, refer to manufacturer's instructions.

Basic Protocol 2: Pyronin Y and Hoechst 33342 Staining for Analyzing Cell Cycle Status

  Material
  • Cells
  • 1× phosphate‐buffered saline (PBS)
  • 70% ethanol (−20°C)
  • FACS buffer (see recipe)
  • Hoechst/PY staining solution (see recipe)
  • Centrifuge
  • Flow cytometer equipped with both 355‐nm UV and 488‐nm blue laser to activate Hoechst 33342 and Pyronin Y (488‐nm laser can be replaced with 532‐nm green or 561‐nm yellow‐green lasers), appropriate filter sets
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Figures

Videos

Literature Cited

Literature Cited
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Internet Resources
  http://www.lifetechnologies.com/us/en/home/life‐science/cell‐analysis/flow‐cytometry/cell‐health‐and‐viability‐assays‐for‐flow‐cytometry/cell‐proliferation‐assays‐for‐flow‐cytometry/click‐it‐edu‐cell‐proliferation‐assay‐kits‐for‐flow‐cytometry.html
  Provides information on Click‐iT Plus Assay Kits.
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