High‐Resolution Cell Cycle and DNA Ploidy Analysis in Tissue Samples

Christina Heinlein1, Daniel Speidel2

1 Children's Medical Research Institute, Westmead, New South Wales, Australia, 2 Sydney Medical School, The University of Sydney, Sydney, New South Wales, Australia
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.39
DOI:  10.1002/0471142956.cy0739s56
Online Posting Date:  April, 2011
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes an easy, rapid, and universal procedure to process fresh and nitrogen‐frozen tissue specimens for high‐resolution cell cycle and DNA ploidy analysis. Unlike other protocols, this procedure does not require treating tissues with enzymes, detergents, or other plasma membrane–lysing chemicals, but it achieves tissue dispersion by a simple two‐step mechanical process that can be performed in ∼5 min. Resulting single‐cell suspensions are fixed with ethanol, stained with propidium iodide, and subjected to flow cytometric DNA content analysis. The method can be applied without any alterations to all tissue types (except bones) derived from several species and results in highly reproducible cell cycle profiles of excellent resolution. The described protocol can be used to reliably and accurately detect subtle cell cycle and ploidy alterations in tissue specimens, including cell cycle arrest, aneuploidy, and apoptosis/necrosis‐associated DNA fragmentation. Curr. Protoc. Cytom. 56:7.39.1‐7.39.11. © 2011 by John Wiley & Sons, Inc.

Keywords: flow cytometry; tissue; DNA content; cell cycle; ploidy; DNA fragmentation; animal models

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

Table of Contents

  • Introduction
  • Basic Protocol 1: High‐Resolution Cell Cycle and Ploidy Analysis in Fresh and Nitrogen‐Frozen Tissue Specimens
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: High‐Resolution Cell Cycle and Ploidy Analysis in Fresh and Nitrogen‐Frozen Tissue Specimens

  Materials
  • Tissue, fresh or snap‐frozen in liquid nitrogen (and stored at −80°C)
  • PBS/EDTA (4°C and room temperature; see recipe)
  • Phosphate‐buffered saline (PBS; MP Biomedicals, cat. no. 2810305 or equivalent)
  • 80% (v/v) ethanol, −20°C
  • Propidium iodide (PI) staining solution with RNase A (see recipe), freshly made
  • 100‐µm nylon cell strainer (BD Falcon, cat. no. 352360 or equivalent)
  • Small tissue culture dish (∼35 mm), the diameter of which is slightly larger than the 100‐µm nylon cell strainer
  • Plunger of a 5‐ml syringe
  • 3‐ml syringes
  • 22‐ and 25‐G needles
  • 35‐µm nylon cell strainer (polystyrene tube with cell‐strainer cap; BD Falcon, cat. no. 352235 or equivalent)
  • 15‐ml polystyrene conical tubes
  • Centrifuge
  • Vortex
  • 1‐ml pipets
  • 37°C water bath
  • Flow cytometer equipped with 488‐nm excitation source and 620‐nm band‐pass filter for PI
  • Appropriate software (e.g., Multicycle; Phoenix Flow Systems)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Burns, E.R., Bagwell, C.B., Hinson, W.G., Pipkin, J.L. Jr., and Hudson, J.L. 1983. Preparation and stability of sixteen murine tissues and organs for flow cytometric cell cycle analysis. Cytometry 4:50‐160.
   Heinlein, C., Deppert, W., Braithwaite, A.W., and Speidel, D. 2010. A rapid and optimization‐free procedure allows the in vivo detection of subtle cell cycle and ploidy alterations in tissues by flow cytometry. Cell Cycle 9:3584‐3590.
   Krishan, A. and Cabana, R. 2004. Flow cytometric analysis of electronic nuclear volume and DNA content in normal mouse tissues. Cell Cycle 3:380‐383.
   Thornthwaite, J.T., Sugarbaker, E.V., and Temple, W.J. 1980. Preparation of tissues for DNA flow cytometric analysis. Cytometry 1:229‐237.
   Vindeløv, L.L., Christensen, I.J., and Nissen, N.I. 1983. A detergent‐trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 3:323‐327.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library