Analysis of Cellular DNA Content by Flow Cytometry

Zbigniew Darzynkiewicz1, Xuan Huang1

1 New York Medical Center, Valhalla, New York
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 5.7
DOI:  10.1002/0471142735.im0507s60
Online Posting Date:  May, 2004
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Abstract

Cellular DNA content can be measured by flow or laser‐scanning cytometry with the aim of (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating the frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing the DNA‐ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and proteolytic treatment to permeabilize cells. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells for subsequent culturing based on DNA‐content differences, is also described. Also presented are methods for staining of cell nuclei isolated from paraffin‐embedded tissues, and deconvolution of DNA‐content‐frequency histograms to estimate the percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content.

Keywords: cell cycle; DNA ploidy; DNA index; Hoechst 33342; propidium iodide; PI; 4′6‐diamidino‐2‐phenylindole; DAPI

     
 
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Table of Contents

  • Basic Protocol 1: Analysis of Fixed Cells Stained with Propidium Iodide
  • Alternate Protocol 1: Analysis of Fixed Cells Stained with 4′,6‐Diamidino‐2‐Phenylindole
  • Basic Protocol 2: DNA Content Analysis Utilizing Detergents and Proteases
  • Alternate Protocol 2: DNA Content Analysis Utilizing Detergents Only
  • Basic Protocol 3: Supravital Staining of DNA
  • Basic Protocol 4: DNA Content Analysis from Paraffin‐Embedded Samples
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

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

  Materials
  • 70% ethanol
  • Cells to be stained
  • PBS ( appendix 2A2)
  • PI/Triton X‐100 staining solution with RNase A (see recipe)
  • 12 × 75–mm centrifuge tubes, polypropylene or silanized preferred
  • Beckman TJ rotor or equivalent
  • Flow cytometer with 488‐nm argon ion laser, or xenon or mercury arc lamp with short‐pass BG12 filter (∼420‐nm cutoff)
  • >600‐nm long‐pass filter (optional)
  • DNA content frequency deconvolution software (e.g., Multicycle from Phoenix Flow Systems, ModFit from Verity Software)

Alternate Protocol 1: Analysis of Fixed Cells Stained with 4′,6‐Diamidino‐2‐Phenylindole

  • DAPI/Triton X‐100 staining solution (see recipe)
  • Flow cytometer with UV light illumination source—e.g., laser tuned to UV light at 340 to 380 nm, or mercury arc lamp with UG1 short‐pass (390‐nm cutoff) excitation and band‐pass 420 ± 20‐nm emission filters

Basic Protocol 2: DNA Content Analysis Utilizing Detergents and Proteases

  Materials
  • Tissue sample (e.g., resected tumor)
  • Citrate/DMSO buffer (see recipe)
  • Internal DNA content standard (see recipe)
  • Cell lysis solution (see recipe)
  • Trypsin‐inactivating solution, ice cold (see recipe)
  • PI/spermidine staining solution, ice cold (see recipe)
  • 0.5 × 25–mm (25‐G × 1‐in.) injection needles
  • 1‐ and 10‐ml disposable syringes
  • 38 × 12.5–mm polypropylene tubes with caps
  • 24‐ to 34‐µm nylon mesh (Small Parts)
  • Flow cytometer with 488‐nm argon ion laser fluorescence excitation source or mercury arc or xenon lamp with BG12 optical filter
  • >600 nm long‐pass filter
  • DNA content frequency deconvolution software (e.g., Multicycle from Phoenix Flow Systems, ModFit from Verity Software)
  • Additional reagents and equipment for counting cells in a hemacytometer ( appendix 3A)

Alternate Protocol 2: DNA Content Analysis Utilizing Detergents Only

  • Cell suspension
  • DAPI/PIPES staining solution (see recipe)
  • Flow cytometer with UV light illumination source (e.g., laser tuned to 340 to 380 nm, mercury arc or xenon lamp with UG1 optical filter)
  • 470 ± 20‐nm band‐pass filter
  • >600‐nm long‐pass filter (for sulforhodamine 101 staining)

Basic Protocol 3: Supravital Staining of DNA

  Materials
  • Hoechst 33342 staining solution (see recipe)
  • Cells in tissue culture medium
  • Flow cytometer with UV light illumination source (e.g., mercury arc or xenon lamp with UG1 optical filter)
  • 470 ± 20‐nm band‐pass filter
NOTE: This protocol is predominantly used for sorting live cells; however, because sensitivity of cells to Hoechst 33342 varies depending on cell type, it is possible that cell viability and their cell cycle progression may be affected by the staining.

Basic Protocol 4: DNA Content Analysis from Paraffin‐Embedded Samples

  Materials
  • Paraffin‐embedded tissue blocks (unit 21.4)
  • Xylene or xylene substitute (e.g., Histo‐Clear, National Diagnostics)
  • 100%, 95%, 80%, 70% and 50% ethanol
  • Protease solution, fresh (see recipe)
  • 0.15 M NaCl
  • DAPI/phosphate staining solution, fresh (see recipe)
  • Microtome (e.g., Shandon Lipshaw)
  • 57‐µm nylon mesh in sheets (for filtering) and made into 2 × 2–cm bags by heat welding (Small Parts)
  • Rotary shaker
  • 1‐ to 2‐mm diameter marble or glass balls
  • 5‐ and 15‐ml tubes
  • 37°C water bath with rotary shaker
  • Phase contrast or interference contrast (Nomarski optics) microscope
  • Flow cytometer with UV light fluorescence excitation source
  • DNA content frequency deconvolution software (e.g., Phoenix Flow Systems Multicycle software)
  • Additional reagents and equipment for hematoxylin and eosin (HE) staining (unit 21.4), light microscopy (unit 21.1), and counting cells in a hemacytometer ( appendix 3A)
CAUTION: Xylene is toxic. Wear gloves and keep lids on jars. When possible, xylene should be substituted by less toxic reagents such as Histo‐Clear.NOTE: Store xylene and ethanol solution in 20‐ml aliquots in closed, xylene‐resistant, glass or plastic containers (e.g., Coplin jars or Erlenmeyer flasks).
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Figures

Videos

Literature Cited

Literature Cited
   Bagwell, B.C. 1993. Theoretical aspects of flow cytometry data analysis. In Clinical Flow Cytometry. Principles and Applications. (K.E. Bauer, R.E. Duque, T.V. Shankey, eds.) pp. 41‐61. Williams & Wilkins, Baltimore.
   Crissman, H.A. and Steinkamp, J.A. 1992. Cytochemical techniques for multivariate analysis of DNA and other cell constituents. In Flow Cytometry and Sorting. (M.R. Melamed, T. Lindmo, and, M.L. Mendelsohn, eds.) pp. 227‐247. Wiley‐Liss, New York.
   Darzynkiewicz, Z., Traganos, F., Kapuscinski, J., Staiano‐Coico, L., and Melamed, M.R. 1984. Accessibility of DNA in situ to various fluorochromes: Relationship to chromatin changes during erythroid differentiation of Friend leukemia cells. Cytometry 5:355‐363.
   Darzynkiewicz, Z., Juan, G., Li, X., Gorczyca, W., Murakami, T., and Traganos, F. 1997. Cytometry in cell necrobiology. Analysis of apoptosis and accidental cell death (necrosis). Cytometry 27:1‐20.
   Darzynkiewicz, Z., Bedner, E., and Traganos, F. 2001. Difficulties and pitfalls in analysis of apoptosis. Meth. Cell Biol. 63:527‐546.
   Darzynkiewicz, Z., Crissman, H., and Jacovverger, J.W. 2004. Cytometry of the cell cycle: Cycling through history. Cytometry 58A: in press.
   Gong, J., Traganos, F., and Darzynkiewicz, Z. 1994. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Anal. Biochem. 218:314‐319.
   Hedley, D.W. 1994. DNA analysis from paraffin‐embedded blocks. Meth. Cell Biol. 41:231‐240.
   Hedley, D.W., Friedlander, M.I., Taylor, I.W., Rugg, C.A., and Musgrove, E. 1983. Method for analysis of cellular DNA content of paraffin‐embedded pathological material using flow cytometry. J. Histochem. Cytochem. 31:1333‐1335.
   Heiden, T., Wang, N., and Tribukait, B. 1991. An improved Hedley method for preparation of paraffin‐embedded tissues for flow cytometric analysis of ploidy and S‐phase. Cytometry 12:614‐621.
   Hitchcock, C.L. and Ensley, J.E. 1993. Technical considerations for dissociation of fresh and archival tumors. In Clinical Flow Cytometry. Principles and Applications. (K.B. Bauer, R.E. Duque, and, T.V. Shankey, eds.) pp. 93‐109.
   Krishan, A. 1987. Effect of drug efflux blockers on vital staining of cellular DNA with Hoechst 33342. Cytometry 8:642‐645.
   Loken, M.M. 1980. Simultaneous quantitation of Hoechst 33342 and immunofluorescence on viable cells using a fluorescence activated cell sorter. Cytometry 1:136‐142.
   Rabinovitch, P.S. 1993. Practical considerations for DNA content and cell cycle analysis. In Clinical Flow Cytometry. Principles and Applications. (K.E. Bauer, R.E. Duque, T.V. Shankey, eds.) pp. 117‐142. Williams & Wilkins, Baltimore.
   Schmid, I. 1999. Assessment of viability, immunofluorescence, and DNA content. Current Protocols in Cytometry 7:11.
   Vindelov, L.L. and Christiansen, I.J. 1994. Detergent and proteolytic enzyme‐based techniques for nuclear isolation and DNA content analysis. Meth. Cell Biol. 41:219‐230.
   Vindelov, 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.
   Wyllie, A.H. 1992. Apoptosis and the regulation of cell numbers in normal and neoplastic tissues. An overview. Cancer Metast. Rev. 11:95‐103.
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