Determining Cell Cycle Stages by Flow Cytometry

Zbigniew Darzynkiewicz1, Gloria Juan1, Elzbieta Bedner1

1 New York Medical College, Valhalla, New York
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 8.4
DOI:  10.1002/0471143030.cb0804s01
Online Posting Date:  May, 2001
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Abstract

This unit describes assays used to determine the distribution of a population of cells to the different stages of the cell cycle as analyzed by flow cytometry. Staining the DNA with different fluorescent dyes, propidium iodide or DAPI, is one of the most direct ways of staging the cells based on DNA content. These staining procedures can be used with fixed cells or detergentā€permeabilized cells. Alternatively, live cells can be stained with the membraneā€permeable stain, Hoechst 33242, and sorted on the basis of DNA content for further culture and characterization. When DNA content measurements are coupled with immunostaining for cyclin expression, it is possible to reliably detect even more stages in progression through the cell cycle.

     
 
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Table of Contents

  • Basic Protocol 1: Cell Cycle Analysis of Fixed Cells Stained with Propidium Iodide
  • Alternate Protocol 1: Cell Cycle Analysis of Fixed Cells Stained with DAPI
  • Basic Protocol 2: Cell Cycle Analysis of Unfixed, Detergent‐Permeabilized Cells Stained with PI
  • Alternate Protocol 2: Cell Cycle Analysis of Unfixed, Detergent‐Permeabilized Cells Stained with DAPI
  • Basic Protocol 3: Staining of Live Cells with Hoechst 33342
  • Basic Protocol 4: Bivariate Analysis of DNA Content and Expression of Cyclins D, E, A, or B1
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Cell Cycle Analysis of Fixed Cells Stained with Propidium Iodide

  Materials
  • Cells to be stained
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Fixative: 70% ethanol
  • Propidium iodide staining solution I (see recipe)
  • Low‐speed centrifuge
  • 12 × 75–mm centrifuge tubes, preferably polypropylene or silanized
  • Flow cytometer with 488‐nm argon‐ion laser fluorescence excitation source
  • Software to deconvolute cellular DNA content frequency histograms (e.g., Multicycle from Phoenix Flow Systems)
  • Additional reagents and equipments for counting and trypsinizing cells (unit 1.1)

Alternate Protocol 1: Cell Cycle Analysis of Fixed Cells Stained with DAPI

  • DAPI staining solution I (see recipe)
  • Flow cytometer with UV illumination source (e.g., mercury‐arc lamp or laser tuned to UV at 340 to 380 nm)

Basic Protocol 2: Cell Cycle Analysis of Unfixed, Detergent‐Permeabilized Cells Stained with PI

  Materials
  • Cells to be stained: 1 × 106 to 5 × 106 cells/ml suspended in PBS ( appendix 2A) or culture medium
  • Propidium iodide staining solution II (see recipe)
  • Flow cytometer with 488‐nm argon‐ion laser fluorescence excitation source
  • Software to deconvolute cellular DNA content frequency histograms (e.g., Multicycle from Phoenix Flow Systems)

Alternate Protocol 2: Cell Cycle Analysis of Unfixed, Detergent‐Permeabilized Cells Stained with DAPI

  • DAPI staining solution II (see recipe)
  • Flow cytometer with UV‐illumination source (e.g., mercury‐arc lamp or laser tuned to UV at 340 to 380 nm)

Basic Protocol 3: Staining of Live Cells with Hoechst 33342

  Materials
  • 1 mg/ml Hoechst 33342 in H 2O (store up to several weeks at 4°C in dark or foil‐wrapped bottles)
  • Cells to be stained: 1 × 106 cells/ml suspended in PBS ( appendix 2A) or culture medium
  • Flow cytometer with UV light illumination source (e.g., mercury‐arc lamp or laser tuned to UV at 340 to 380 nm)

Basic Protocol 4: Bivariate Analysis of DNA Content and Expression of Cyclins D, E, A, or B1

  Materials
  • Cells to be analyzed
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Fixative: 80% ethanol or absolute methanol, −20°C.
  • 0.25% (v/v) Triton X‐100 in PBS, pH 7.4 (store at 4°C)
  • Rinsing buffer: 1% (w/v) bovine serum albumin (BSA) in PBS, pH 7.4 (store at 4°C)
  • Anti‐cyclin IgG1 antibodies: e.g., mouse monoclonal antibodies to cyclin B1 (clone GNS‐1), cyclin A (clone BF‐683), cyclin D1 (clone G124‐326), cyclin D3 (clone G107‐565), and cyclin E (clone HE12); all provided by PharMingen; antibodies to cyclin D1 may also be obtained from Immunotech/Coulter
  • Mouse IgG1 (isotypic control)
  • FITC‐conjugated goat anti–mouse IgG
  • Propidium iodide staining solution III (see recipe)
  • 15‐ml conical tubes, polypropylene or silanized
  • Low‐speed centrifuge
  • Flow cytometer equipped with 488‐nm argon laser fluorescence excitation source
  • Additional reagents and equipment for collecting and preparing cells for fixation (see protocol 1, step , b, or c)
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Figures

Videos

Literature Cited

Literature Cited
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