Analysis of DNA Content and Green Fluorescent Protein Expression

Ingrid Schmid1, Kathleen M. Sakamoto1

1 UCLA School of Medicine, Los Angeles, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.16
DOI:  10.1002/0471142956.cy0716s16
Online Posting Date:  May, 2001
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Abstract

Green fluorescent protein (GFP) is an intracellular reporter molecule widely utilized for assessment of gene transfer and expression. Enhanced variants have been cloned into various expression vectors suited for many different cell types. To study the effect of a gene of interest on cell cycle progression, it is desirable to measure GFP expression in combination with DNA content. This approach is difficult, as most suitable fluorescent DNA dyes are too large to pass through intact cell membranes, but permeabilization will allow GFP to leak out. The authors present a protocol with a cell preparation technique designed to maintain the delicate balance between retaining GFP fluorescence and obtaining adequate DNA histogram resolution. An describes a combined GFP fluorescence and cell cycle analysis using unpermeabilized cells stained with the vital dye Hoechst 33342.

     
 
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Table of Contents

  • Basic Protocol 1: DNA Content Analysis in Combination with Assessment of Red‐Shifted Green Fluorescent Protein Expression in Fixed/Permeabilized Cells
  • Alternate Protocol 1: DNA Content Analysis in Combination with Assessment of Red‐shifted Green Fluorescent Protein Expression in Unfixed Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: DNA Content Analysis in Combination with Assessment of Red‐Shifted Green Fluorescent Protein Expression in Fixed/Permeabilized Cells

  Materials
  • Cells
  • Phosphate buffered saline (PBS; appendix 2A), ∼4°C
  • Fixation solution, ∼4°C (see recipe)
  • 70% ethanol, −20°C
  • recipePropidium iodide (PI) working solution (see recipe)
  • Centrifuge, 2° to 8°C
  • 37°C water bath
  • 70‐µm nylon mesh (e.g., Fisher Scientific's Spectra/Mesh N, Becton Dickinson's Falcon Nylon Cell Strainers)
  • Flow cytometer with 488‐nm excitation and 530/30 and 585/42 nm band‐pass filters or equivalent
  • Additional reagents and equipment for counting cells ( appendix 2A) and retroviral transduction of red‐shifted GFP into cells (unit 9.12).

Alternate Protocol 1: DNA Content Analysis in Combination with Assessment of Red‐shifted Green Fluorescent Protein Expression in Unfixed Cells

  • Culture medium, 37°C
  • 1 mg/ml Hoechst 33342 stock solution (see recipe)
  • Flow cytometer with 488‐nm and 325‐nm excitation and 530/30 and 424/44 nm band‐pass filters or equivalent
  • Additional reagents and equipment for concurrent dead cell discrimination (unit 9.2; optional).
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Figures

Videos

Literature Cited

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Key Reference
  Chu et al. 1999. See above.
  Describes the procedure presented in the .
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