LSC Application: Comet Assay Analysis of DNA Damage

The single-cell gel electrophoresis (SCGE) or comet assay is a very sensitive method for measuring DNA strand breaks in individual cells. The comet assay is widely used in environmental toxicology, cancer research, and radiation biology to assess DNA damage. A small number of cells are embedded in agarose gel on a microscope slide, lysed, electrophoresed, and stained with fluorescent DNA binding dye.

Damaged DNA migrates during electrophoresis from the nucleus towards the anode, forming a shape of a “comet” with a head (cell nucleus with intact DNA) and a tail (relaxed and broken DNA). We use the LSC^® for automatic analysis of the comet assay. The percentage of events lying in the sub-G₁ area of the DNA histogram is a measure of DNA damage.

The dose-dependent increase in DNA damage is mainly seen as an increase in the number of events in the far sub-G₁ area with low values of PI integral fluorescence and decreasing values of max pixel. The dot plot and matching histogram represents DNA damage in HaCaT cells irradiated with 47 kJ/m² UVA. Fluorescence photo of comets representing the events in the far sub-G₁ area with low values of PI integral fluorescence.

Sample Preparation
HaCaT cells (300,000 cells/ml) were embedded in 1% low melting agarose in PBS without Ca²⁺ and Mg²⁺ by a three layer sandwich technique (1) on top of custom frosted microscope slides with a clear rectangular window section (2). DNA damage was introduced by UVA irradiation. The cells were immediately lysed in cold lysis solution (2.5 M NaCl, 100 mM EDTA, 1% N-laurylsarcocine, 10 mM Tris-base, pH 10, with 10% DMSO and 1% Triton X-100) for one hour at 4 °C. The DNA was allowed to unwind for 20 min. in electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH 13.2) at 4°C and electrophoresed at 20 V and 300 mA for 20 min at 4°C. The gels were washed in neutralizing buffer (400 mM Tris-HCl, pH 7.5) three times for 5 min. DNA was stained with propidium iodide (PI).

LSC Analysis
The embedded cells were focused on and scanned on the central portion of the slide window to avoid possible edge artifacts. The scanning was run on an LSC equipped with a BX50 Olympus microscope and a 488-nm argon laser, using a 20X dry objective. The sensitivity threshold was set to contour comet heads only. PI fluorescence was integrated from a region six pixels broader than the threshold contour around the comet head. One thousand events were scanned per slide. The G₁ phase peak of the integrated fluorescence histogram was normalized to DNA index = 1. After electrophoresis, cells with damaged DNA contained less DNA than did the cells in the G₁ phase and therefore appeared in the sub-G₁ area of the histogram. Software statistics provide the percentage of cells in a predefined sub-G₁ area, quantifying the percentage of DNA damage in a cell population. The percentage of cells in the sub-G₁ area is a measure of DNA damage. There is a dose-dependent increase in the percentage of events in the sub-G₁ area in UVA irradiated HaCaT cells. In addition, the imaging function of the LSC makes it possible to obtain digital images of comets by rescanning the microscope slide. The LSC results compare well with tail moment analysis from comet images (3).

(Procedure and data from Anita B. Petersen, Robert Gniadecki, Hans Christian Wulf, Department of Dermatology D92, Bispebjerg Hospital, Copenhagen, Denmark)

References
1. Piperakis SM, Visvardis E-E, and Tassiou (1999). Comet assay for nuclear DNA damage. Methods Enzymol. 300:184 -194.
2. Smith MJ and O`Neill KL (1998). Microscope slide for enhanced analysis of DNA damage using the comet assay. Biotechniques 25:49-50.
3. Petersen AB, Gniadecki R and Wulf HC (2000). Laser scanning cytometry for comet assay analysis. Cytometry 39:10-15.