Methods for Synchronizing Cells at Specific Stages of the Cell Cycle

Joany Jackman1, Patrick M. O'Connor2

1 U.S.A.M.R.I.I.D., Fort Dietrick, Maryland, 2 Agouron Pharmaceuticals, San Diego, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 8.3
DOI:  10.1002/0471143030.cb0803s00
Online Posting Date:  May, 2001
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Abstract

Exponentially growing cells are asynchronous with respect to the cell cycle stage. Detection of cell cycle‐related events is improved by enriching the culture for cells at the stage during which the particular event occurs. Methods for synchronizing cells are provided here, including those based on morphological features of the cell (mitotic shake‐off), cellular metabolism (thymidine inhibition, isoleucine depravation), and chemical inhibitors of cell progression in G1 (lovastatin), S (aphidicolin, mimosine), and G2/M (nocodazole). Applications of these methods and the advantages and disadvantages of each are described.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Enrichment of Mitotic Cells by Mitotic Shake‐Off
  • Alternate Protocol 1: Pre‐Enrichment of Exponential Cultures for Mitotic Cells
  • Alternate Protocol 2: Enrichment of Mitotic Cells by Nocodazole Arrest
  • Basic Protocol 2: Enrichment of Cells at G0/G1 by Serum Starvation
  • Alternate Protocol 3: Enrichment of Cells at G0/G1 by Amino Acid Starvation
  • Basic Protocol 3: Enrichment of G1‐Phase Cells Using Lovastatin
  • Alternate Protocol 4: Enrichment of G1‐Phase Cells by Mimosine Arrest
  • Basic Protocol 4: Synchronizing Cells at the Onset of S Phase by Double‐Thymidine Block
  • Alternate Protocol 5: Performing Sequential G1/S Blocks
  • Support Protocol 1: Determining Mitotic Index
  • Support Protocol 2: Monitoring [3H]Thymidine Incorporation into DNA by TCA Precipitation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Enrichment of Mitotic Cells by Mitotic Shake‐Off

  Materials
  • Human colon carcinoma cell line (HT‐29 cells), grown in 162‐cm2 tissue culture flasks
  • RPMI‐15: RPMI 1640 supplemented with 15% (v/v) heat‐inactivated fetal bovine serum (FBS; appendix 2A), prewarmed to 37°C
  • RPMI 1640 medium ( appendix 2B), prewarmed to 37°C
  • 0.5× trypsin solution (see recipe)
  • Additional reagents and equipment for trypsinizing and counting cells (unit 1.1)

Alternate Protocol 1: Pre‐Enrichment of Exponential Cultures for Mitotic Cells

  Materials
  • CA46 cells, grown in 75‐cm2 tissue culture flasks
  • 0.4 mg/ml nocodazole (diluted from 4 mg/ml nocodazole; see recipe)
  • RPMI‐15: RPMI 1640 supplemented with 15% heat‐inactivated FBS ( appendix 2A), prewarmed to 37°C
  • Additional reagents and equipment for counting cells (unit 1.1)

Alternate Protocol 2: Enrichment of Mitotic Cells by Nocodazole Arrest

  Materials
  • NIH‐3T3 cells, grown in 100‐mm tissue culture plates
  • 1× trypsin (Life Technologies)
  • DMEM ( appendix 2A), prewarmed to 37°C
  • DMEM‐0.5: DMEM supplemented with 0.5% (v/v) heat‐inactivated FBS ( appendix 2A), prewarmed to 37°C
  • DMEM‐10: DMEM supplemented with 10% (v/v) heat‐inactivated FBS, prewarmed to 37°C
  • Additional reagents and equipment for trypsinizing and counting cells (unit 1.1)

Basic Protocol 2: Enrichment of Cells at G0/G1 by Serum Starvation

  Materials
  • CA46 cells, grown in 162‐cm2 tissue culture flasks
  • Isoleucine‐free minimal essential medium (e.g., Select‐Amine Minimal Medium, Life Technologies), prewarmed to 37°C
  • Dialyzed FBS (Life Technologies)
  • RPMI‐15: RPMI 1640 supplemented with 15% (v/v) heat‐inactivated FBS ( appendix 2A)

Alternate Protocol 3: Enrichment of Cells at G0/G1 by Amino Acid Starvation

  Materials
  • MCF‐7 cells, grown in 100‐mm tissue culture plates
  • 1× trypsin (Life Technologies)
  • DMEM‐10: DMEM supplemented with 10% (v/v) heat‐inactivated FBS ( appendix 2A), prewarmed to 37°C
  • 10 mM activated lovastatin (see recipe)
  • DMEM‐10 containing 5 mM mevalonate (Sigma)
  • Additional reagents and equipment for trypsinizing and counting cells (unit 1.1)

Basic Protocol 3: Enrichment of G1‐Phase Cells Using Lovastatin

  Materials
  • CA46 cells, grown in 75‐cm2 tissue culture flasks
  • RPMI‐15/mimosine: RPMI supplemented with 15% (v/v) heat‐inactivated FBS ( appendix 2A) and 400 µM mimosine (diluted from 100 mM mimosine; see recipe), prewarmed to 37°C
  • RPMI‐15: RPMI supplemented with 15% (v/v) heat‐inactivated FBS, prewarmed to 37°C

Alternate Protocol 4: Enrichment of G1‐Phase Cells by Mimosine Arrest

  Materials
  • HeLa cells, grown in 162‐cm2 tissue culture flasks
  • DMEM‐10/thymidine: DMEM supplemented with 10% (v/v) heat‐inactivated FBS ( appendix 2A) and 2 mM thymidine (diluted from 100 mM thymidine; see recipe), prewarmed to 37°C
  • DMEM, prewarmed to 37°C
  • DMEM‐10: DMEM supplemented with 10% (v/v) heat‐inactivated FBS ( appendix 2A), prewarmed to 37°C
  • 1× trypsin (Life Technologies)
  • 1× PBS, pH 7.4 ( appendix 2A)
  • Additional reagents and equipment for trypsinizing and coating cells (unit 1.1)

Basic Protocol 4: Synchronizing Cells at the Onset of S Phase by Double‐Thymidine Block

  Materials
  • Burkitt's lymphoma cell line (CA46 cells), grown in RPMI‐15, synchronized and unsynchronized
  • RPMI‐15: RPMI supplemented with 15% (v/v) heat‐inactivated FBS
  • 1× PBS, pH 7.4 ( appendix 2A), ice cold
  • 0.5× PBS, pH 7.4 ( appendix 2A), or 75 mM KCl, ice cold
  • 3:1 (v/v) ethanol/glacial acetic acid, ice cold
  • Giemsa stain (Sigma, Life Technologies; optional)

Alternate Protocol 5: Performing Sequential G1/S Blocks

  Materials
  • CA46 cells, released from synchronization
  • RPMI‐15: RPMI supplemented with 15% (v/v) heat‐inactivated FBS ( appendix 2A), prewarmed to 37°C
  • [3H]thymidine (20 to 60 Ci/mmol)
  • 1× PBS ( appendix 2A)
  • 0.5× PBS ( appendix 2A)
  • 20% (v/v) TCA, ice cold
  • 5% (v/v) TCA, ice cold
  • 95% ethanol
  • Dry ice/methanol bath
  • Wet ice in bucket
  • Glass fiber filters
  • Vacuum manifold
  • Vacuum drying oven, 80°C
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Figures

Videos

Literature Cited

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