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, null
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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Abstract

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

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

Basic Protocol: Preparation and DRAQ5 Staining of Live Cells for Analysis Using Flow Cytometry or Imaging

 Materials
  • 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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Figures

  •  FigureFigure 7.25.1 Flow cytometric analysis of the rapid cellular uptake of DRAQ5, demonstrating the ability to discriminate DNA content using either (A) 488-nm excitation (emission wavelength [Em] >715 nm) or (B) 633-nm excitation (Em >695 nm). DRAQ5-DNA fluorescence intensity distributions (for each channel number) of live human B cell SU-DHL-4 lymphoma cells were monitored during 40-sec acquisition periods (~8 × 103 events) at each of the indicated periods of exposure to the dye. Cell suspensions (4 × 105 cells/ml) were prepared in complete medium supplemented with 10 mM HEPES at 37°C. The dye was added immediately prior to analysis at a concentration of 20 µM DRAQ5. Data show the rapid establishment of distributions for content analysis and the similarity of the profiles for blue- or red-line excitation.
    View Image
  •  FigureFigure 7.25.2 Comparison of the DNA content distributions derived from live (solid line) and ethanol-fixed (dashed line) human B cell SU-DHL-4 lymphoma cells (4 × 105 cells/ml). Live cells were stained as described for Figure 7.25.1 and data were acquired using 488-nm excitation (>715-nm emission) after a 560-sec exposure to 20 µM DRAQ5. Fixed cells were prepared by resuspending washed live-cell pellets in 70% (v/v) ethanol in PBS and holding 30 min on ice prior to centrifugation, washing, and resuspension in PBS supplemented with 20 µM DRAQ5. Data were acquired as for live cells. The coefficient of variation (CV) for the G1 peak is <5%.
    View Image
  •  FigureFigure 7.25.3 Confocal microscopy of DRAQ5-stained live cells. (A) Single-plane confocal fluorescence image and (B) corresponding transmission image of live MCF-7 breast tumor cells in a microcolony (Ex 647 nm; Em 680/32 nm) showing DRAQ5-stained nuclei (10 µM) and revealing nuclear architecture. Image captured 180 sec after dye addition to the culture medium containing 10 mM HEPES, pH 7.2. (C) Live human osteosarcoma cells (U2-OS) similarly stained with 20 µM DRAQ5 (600 sec), revealing nuclear features and an anaphase cell (arrow).
    View Image
  •  FigureFigure 7.25.4 Detection of intracellular malarial parasite forms in artificially infected blood cultures using DRAQ5. (A) Fluorescence and (B) transmission images of an air-dried unfixed blood film mounted directly in PBS supplemented with 20 µM DRAQ5. The fluorescence image shows bright regions in red blood cells, representing developmental forms of Plasmodium falciparum [NF54]. Arrows indicate an early marginal form (mf) and a later trophozoite (tr) form. Images were obtained using a Bio-Rad 1024MP confocal imaging system (647-nm excitation). Blood films were kindly supplied by Dr. Laurent Rénia (Cochin, Gustave Roussy, Paris).
    View Image
  •  FigureFigure 7.25.5 Live Metarhizium anisopliae mycelium mounted 30 min in 20 µM DRAQ5 in PBS. (A) Fluorescence and (B) reflectance images were obtained using a Bio-Rad 1024MP confocal imaging system (combined 647- and 568-nm laser excitation). Fungal nuclei (arrows) are shown. Cultures were kindly supplied by Dr. Tariq M. Butt (School of Biological Sciences, University of Wales, Swansea, United Kingdom).
    View Image

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Literature Cited

Literature Cited
    Albota, M., Xu, C., and Webb, W. 1998. Two-photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm. Appl. Opt. 37:7352-7356.
    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.
    Fitzgerald, K.A., Rowe, D.C., Barnes, B.J., Caffrey, D.R., Visintin, A., Latz, E., Monks, B., Pitha, P.M., and Golenbock, D.T. 2003. LPS-TLR4 signaling to IRF-3/7 and NF-B involves the toll adapters TRAM and TRIF. J. Exp. Med. 198:1043-1055.
    Fox, M.E. and Smith, P.J. 1995. Subcellular localisation of the antitumour drug mitoxantrone and the induction of DNA damage in resistant and sensitive human colon carcinoma cells. Cancer Chemother. Pharmacol. 35:403-410.
    Frey, T. 1995. Nucleic acid dyes for detection of apoptosis in live cells. Cytometry 21:265-274.
    Frey, T., Yue, S., and Haugland, R.P. 1995. Dyes providing increased sensitivity in flow-cytometric dye-efflux assays for multidrug resistance. Cytometry 20:218-227.
    Latt, S.A. and Langlois, R.G. 1990. Fluorescent probes of DNA microstructure and DNA synthesis. In Flow Cytometry and Sorting. 2nd ed. (M.R. Melamed, T. Lindmo, and, M.L. Mendelsohn, eds.) pp. 249-290. Wiley-Liss, New York.
    Lown, J.W., Morgan, A.R., Yen, S.-F., Wang, Y.H., and Wilson, W.D. 1985. Characteristics of the binding of the anticancer agents mitoxantrone and ametantrone and related structures to deoxyribonucleic acids. Biochemistry 24:4028-4035.
    Mitta, B., Rimann, M., Ehrengruber, M.U., Ehrbar, M., Djonov, V., Kelm, J., and Fussenegger, M. 2002. Advanced modular self-inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction. Nucleic Acids Res. 30:e113.
    Plander, M., Brockhoff, G., Barlage, S., Schwarz, S., Rothe, G., and Knuechel, R. 2003. Optimization of three- and four-color multiparameter DNA analysis in lymphoma specimens. Cytometry 54A:66-74.
    Schmitt, A., Gutierrez, G.J., Lenart, P., Ellenberg, J., and Nebreda, A.R. 2002. Histone H3 phosphorylation during Xenopus oocyte maturation: Regulation by the MAP kinase/p90Rsk pathway and uncoupling from DNA condensation. FEBS Lett. 518:23-28.
    Smith, P.J., Morgan, S.A., and Watson, J.V. 1991. Detection of multidrug resistance and quantification of responses of human tumour cells to cytotoxic agents using flow cytometric spectral shift analysis of Hoechst 33,342-DNA fluorescence. Cancer Chemother. Pharmacol. 27:445-450.
    Smith, P.J., Blunt, N.J., Desnoyers, R., Giles, Y., and Patterson, L.H. 1997a. DNA topoisomerase II-dependent cytotoxicity of alkylaminoanthraquinones and their N-oxides. Cancer Chemother. Pharmacol. 39:455-461.
    Smith, P.J., Desnoyers, R., Patterson, L.H., and Watson, J.V. 1997b. Flow cytometric analysis and confocal imaging of anticancer alkylaminoanthraquinones and their N-oxides in intact human cells using 647-nm krypton laser excitation. Cytometry 27:43-53.
    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.
    Smith, P.J., Blunt, N., Wiltshire, M., Hoy, T., Teesdale-Spittle, P., Craven, M.R., Watson, J.V., Amos, W.B., Errington, R.J., and Patterson, L.H. 2000. Characteristics of a novel deep red/infrared fluorescent cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy. Cytometry 40:280-291.
    Smith, P.J., Wiltshire, M., Davies, S., Chin, S.-F., Campbell, A.K., and Errington, R.J. 2002. DNA damage-induced [Zn(2+)](i) transients: Correlation with cell cycle arrest and apoptosis in lymphoma cells. Am. J. Physiol. Cell Physiol. 283:C609-C622.
    Snyder, D.S. and Garon, C.F. 2003. Decreased uptake of bodipy-labelled compounds in the presence of the nuclear stain, DRAQ5. J. Microsc. 211:208-211.
    Snyder, D.S. and Small, P.L. 2001. Staining of cellular mitochondria with LDS-751. J. Immunol. Methods 257:35-40.
    Terstappen, L.W., Shah, V.O., Conrad, M.P., Recktenwald, D., and Loken, M.R. 1988. Discriminating between damaged and intact cells in fixed flow cytometric samples. Cytometry 9:477-484.
    Terstappen, L.W., Meiners, H., and Loken, M.R. 1989. A rapid sample preparation technique for flow cytometric analysis of immunofluorescence allowing absolute enumeration of cell subpopulations. J. Immunol. Methods 123:103-112.
    Van Zandvoort, M.A., de Grauw, C.J., Gerritsen, H.C., Broers, J.L., oude Egbrink, M.G., Ramaekers, F.C., and Slaaf, D.W. 2002. Discrimination of DNA and RNA in cells by a vital fluorescent probe: Lifetime imaging of SYTO13 in healthy and apoptotic cells. Cytometry 47:226-235.
    Waggoner, A.S. 1990. Fluorescent probes for cytometry. In Flow Cytometry and Sorting. 2nd ed. (M.R. Melamed, T. Lindmo, and, M.L. Mendelsohn, eds.) pp. 209-225. Wiley-Liss, New York.
    Weng, A.P., Nam, Y., Wolfe, M.S., Pear, W.S., Griffin, J.D., Blacklow, S.C., and Aster, J.C. 2003. Growth suppression of pre-T acute lymphoblastic leukemia cells by inhibition of notch signaling. Mol. Cell. Biol. 23:655-664.
    Wiltshire, M., Patterson, L.H., and Smith, P.J. 2000. A novel deep red/low infrared fluorescent flow cytometric probe, DRAQ5NO, for the discrimination of intact nucleated cells in apoptotic cell populations. Cytometry 39:217-223.
 Internet Resources
    http://www.biostatus.co.uk/draq5_msds.html

This material safety data sheet provides health and safety information for DRAQ5.

    http://www.cx.unibe.ch/dkf7/applications.html

The Flow-Cytometry Laboratory of the Department of Clinical Research, University of Bern, has provided a protocol for immunophenotyping that includes DRAQ5 staining.

    http://www.norakbioscience.com/PDF/AZSBS2003.pdf
    http://www.norakbioscience.com/PDF/SBS03Transfluor_Alpha.pdf

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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