DRAQ5 Labeling of Nuclear DNA in Live and Fixed Cells

Paul J. Smith1, Marie Wiltshire1, Rachel J. Errington1

1 University of Wales College of Medicine, Heath Park, Cardiff
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.25
DOI:  10.1002/0471142956.cy0725s28
Online Posting Date:  May, 2004
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This unit describes the use of a novel DNA‐detecting far‐red‐fluorescing dye, DRAQ5, a modified anthraquinone, which has a unique combination of properties exploitable by cytometry. These include a high capacity to permeate the cell membrane, a high DNA binding affinity and selectivity, a fluorescence emission spectrum beyond that of fluorescein, phycoerythrin, Texas Red, Cy3, and EGFP, and excitation characteristics separate from those of propidium iodide. In this unit, methods are presented for preparation and analysis of both live and fixed cells stained with DRAQ5. While the focus is on flow cytometric assays, typical imaging applications are also indicated because the staining protocols share the same essential features.

Keywords: anthraquinone; intercalation; DNA staining

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Table of Contents

  • Basic Protocol 1: Preparation and DRAQ5 Staining of Live Cells for Analysis Using Flow Cytometry or Imaging
  • Alternate Protocol 1: DRAQ5 Staining of Fixed Cells
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation and DRAQ5 Staining of Live Cells for Analysis Using Flow Cytometry or Imaging

  • Mammalian cells of interest to be grown attached or in suspension
  • Complete medium appropriate for cells of interest, with optional 10 mM HEPES (e.g., Sigma), pH 7.2
  • 5 mM DRAQ5 acidified stock (Biostatus); store at room temperature or 4°C
  • Flow cytometer (e.g., FACS Vantage cell sorter; Becton Dickinson) with two lasers emitting at 488 nm and 633 nm, respectively, and appropriate software (e.g., CellQuest; Becton Dickinson) or laser scanning microscope, such as 1024MP scanning unit with LaserSharp software (Bio‐Rad Laboratories) attached to a Zeiss Axiovert 135 (Carl Zeiss) with 63× 1.4–numerical aperture (NA) or 40× 1.3‐NA oil‐immersion lens, operating in confocal laser scanning microscope (CLSM) mode using 488‐, 568‐, or 647‐nm lines of krypton‐argon laser
  • Additional reagents and equipment for cell culture and detachment of adherent cells
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Literature Cited

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   Allman, R., Errington, R.J., and Smith, P.J. 2003. Delayed expression of apoptosis in human lymphoma cells undergoing low‐dose taxol‐induced mitotic stress. J. Cancer 88:1649‐1658.
   Bell, D.H. 1988. Characterization of the fluorescence of the antitumor agent, mitoxantrone. Biochim. Biophys. Acta 949:132‐137.
   Bestvater, F., Spiess, E., Strobrawa, G., Hacker, M., Fuerer, T., Porwol, T., Berchner‐Pfannschmidt, U., Wotzlaw, C., and Acker, H. 2002. Two‐photon fluorescence absorption and emission spectra of dyes relevant for cell imaging. J. Microsc. 208:108‐115.
   Darzynkiewicz, Z. and Kapuscinski, J. 1990. Acri‐dine orange: A versatile probe of nucleic acids and other cellular constituents. In Flow Cytometry and Sorting. 2nd ed. (M.R. Melamed, T. Lindmo, and, M.L. Mendelsohn, eds.) pp. 291‐314. Wiley‐Liss, New York.
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   Smith, P.J., Wiltshire, M., Davies, S., Patterson, L.H., and Hoy, T. 1999. A novel cell permeant and far red‐fluorescing DNA probe, DRAQ5, for blood cell discrimination by flow cytometry. J. Immunol. Methods 229:131‐139.
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Internet Resources
  This material safety data sheet provides health and safety information for DRAQ5.
  The Flow‐Cytometry Laboratory of the Department of Clinical Research, University of Bern, has provided a protocol for immunophenotyping that includes DRAQ5 staining.
  These sites provide examples of fix‐and‐stain protocols using DRAQ5.
  Research supported by the UK Research Councils' Basic Technology Research Programme Grant GR/S23483 and UK Biotechnology and Biological Sciences Research Council Grant SBRI19666.
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