Journal of Histochemistry and Cytochemistry, Vol. 49, 921-922, July 2001, Copyright © 2001, The Histochemical Society, Inc.
Differential Staining of DNA Strand Breaks in Dried Comet Assay Slides
L. BenítezBribiescaa,
P. Sáncheza,
J. Toledoa,
R. Peñarrojaa,
M. Floresa, and
J. Sosab
a Oncological Research Unit, National Medical Center S-XXI, México, DF
b Neurological Research Unit, National Medical Center S-XXI, México, DF
Correspondence to:
L. BenítezBribiesca, Oncological Research Unit, National Medical Center SXXI, IMSS, Av. Cuauhtemoc 330, Col. Doctores DF 06720, Mexico. E-mail: luisbenbri@mexis.com
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Summary |
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The comet assay involves embedding cells in agarose on microscope slides. After lysis and electrophoresis, staining is usually performed with a fluorescent DNA-binding dye and observation is carried out on fresh wet slides through an epifluorescence microscope. We present here a simple alternative for preservation of the agarose comet slides and a fluorescent staining that allows fine differential analysis of DNA strand breaks under confocal microscopy. Lymphocytes were processed according to previous published methods. Slides were quickly dehydrated in a hot oven at 50C for 20 min. Once the agarose layer was dried and reduced to a thin film, slides were treated with RNase. Image analysis showed higher tail length, total area, and tail moment. Using confocal microscopic optical sectioning, a thickness of approximately 180 µm for wet slides and 12 µm for dehydrated gels was calculated. Acridine orange, used for DNA differential staining, allowed quantitation of metachromasia and orthochromasia with confocal scanning microscopy. Differences between alkaline and neutral comet assay with AO were clear-cut and, in principle, a metachromatic index can be calculated. (J Histochem Cytochem 49:921922, 2001)
Key Words:
comet assay, DNA strand breaks, acridine orange, metachromasia
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Introduction |
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Single-cell gel electrophoresis (SCGE), also known as the comet assay, is already recognized as one of the most sensitive methods for measuring DNA strand breaks. The method is simple, rapid, and allows observations at the level of single cells (Collins et al. 1997
). Since its original description by Ostling and Johanson 1994
, the technique has undergone a number of modifications that have enhanced its versatility and applications. Many comprehensive reviews covering the different methods and applications have been published (Fairbairn et al. 1995
; Anderson et al. 1998
; Tice et al. 2000
). The basis of the comet assay involves embedding cells in low melting-point agarose on microscope slides. After lysis and electrophoresis, staining is usually performed with a fluorescent DNA-binding dye and observation is carried out on fresh wet slides through an epifluorescence microscope. Wet agarose slides pose two problems. First, they are not permanent, and second, because of the thickness of the agarose layer, fine microscopic focusing is difficult to achieve. Several authors have tried different procedures to preserve slides from comet assays using various fixatives, drying methods, and stains (Woods et al. 1999
). We present here a simple alternative for preservation of SCGE slides and a fluorescent staining that allows fine differential analysis of strand breaks under confocal microscopy.
Peripheral blood lymphocytes obtained from healthy blood donors were resuspended in PBS buffer and adjusted at 1 x 106 cells/ml. They were subjected to oxidative damage with different concentrations of hydrogen peroxide (100 at 400 µM in PBS for 5 min at 4C). After washing of cells in PBS, they were tested for viability using the tryptan blue 0.05% exclusion test. Lymphocytes were then processed according to two different methods. Briefly, cell suspensions adjusted at 104 cells were embedded in 1% normal melting point agarose at 40C. The content was immediately pipeted onto frosted microscope slides and allowed to gel. For alkaline assay lysis, a solution containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris adjusted at pH 10, plus 1% Triton X-100 was used at 4C for 1 hr. Then slides were placed for 20 min in the electrophoresis buffer containing 0.3 N NaOH, 1 mM EDTA, pH 10, to allow unwinding of DNA (Collins et al. 1993
). Other sets were lysed with the same solutions but adjusting pH to 13 (Tice et al. 2000
). Electrophoresis was performed at 0.78 V/cm on ice for 20 min. Slides were then washed in 0.4 M Tris buffer, pH 7.5, for 15 min. For the neutral non-denaturing comet assay (Olive et al. 1990
), the lysis solution contained 100 mM NaCl, 10 mM Tris buffer, pH 8.3, and the agarose slides were immersed on this solution for 1 hr at room temperature (RT). After washing, slides were electrophoresed at 1 V/cm for 20 min in 90 mM Tris, 2 mM EDTA buffer, pH 8.3. Slides were washed in 0.4 M Tris buffer, pH 7.5, for 10 min. One set of slides was left in a humid chamber before observation. The other set was quickly dehydrated in a hot oven at 50C for 20 min. Once the agarose layer was completely dry and reduced to a thin film, slides were incubated in RNase (100 µM/ml) for 30 min at RT. After fixation in alcoholacetone (v/v) for 1 min and immersion in 0.1 N HCl for 5 min, they were washed in veronal acetate buffer, pH 3.0, and then stained with acridine orange (AO), 10 µg/ml in veronalacetate buffer, pH 4.5. For image analysis, randomly selected comets were digitalized and analyzed with a commercially available program for comet assay (PC Image CASyS; Comet Analysis Synoptics, Cambridge, UK). Slides were also examined by confocal laser scanning microscopy in a Zeiss LSM-510 equipped with Ar-He-Ne lasers. Images were taken using simultaneous dual-channel scanning and were transformed into three-dimensional views using optical sections taken at different intervals. Comets in dried gels are easily focused both in a regular epifluorescence microscope and in confocal analysis. Conversely, wet gels show a diffuse image and superimposition of cells in different planes. Image analysis showed a higher value for tail length, total area, and tail moment in dehydrated slides. Confocal microscopic analysis allowed a precise estimation of gel thickness, showing that wet slides are approximately 180 µm thick, whereas dehydrated gels are approximately 12 µm thick. Acridine orange intercalates into double-stranded nucleic acids or may bind electrostatically to single-stranded regions under specific conditions of pH and ionic strength. In the first type of binding, a green orthochromatic fluorescence is obtained, whereas in the second a metachromatic fluorescence is produced (Darzynkiewicz et al. 1979
) (Fig 1). Because RNA produces AO metachromasia, it is important to treat slides with RNase. Analysis at the red and green wavelengths allowed quantitation of metachromatic and orthochromatic DNA fragments presenting various molecular conformations of DNA in the alkaline and neutral comet assay. In principle, a metachromatic index can be calculated for each assay that should show the relative amount of single and double DNA strand breaks. The technical modification proposed here permits semipermanent slides to be obtained that allow fine focusing for image analysis. Staining with acid AO offers the possibility to disclose and quantitate different molecular conformations of DNA strand breaks in the comet assay.

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Figure 1.
A typical comet from a dry gel slide stained with acid acridine orange. Confocal laser microscopic analysis at different wavelengths: left, green orthochromasia; middle, red metachromasia; right, combined image.
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Acknowledgments |
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Supported by CONACYT, grant #25781-M.
Received for publication December 12, 2000; accepted February 16, 2001.
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