The comet assay

This assay was conducted at Stellenbosch University, South Africa, under the supervision of Professor Sophie Reinecke.

Principles behind the method The assay used for these experiments was developed by Singh et al. (1988), as a modication of the technique introduced by Østling and Jo-hanson (1984). Individual cells are incorporated into an agarose gel, which, after lysing of the cells and unwinding of their DNA, is electrophoresed in an alkaline solution. The gels are rinsed and dried, whereupon the cells are dyed and investigated under a uorescence microscope. If the DNA of a cell is heavily damaged, i.e. if there are many DNA strand breaks, the electrophoresis current will drag the loose ends and fragments out of the nucleus in the direction of the current. In the microscope, the cell takes on a comet-like appearance, which is why the name `comet assay' often is used for this technique, even though it formally is referred to as the Single Cell Gel Electrophoresis technique. In an undamaged cell, however, the DNA will stay mostly in the nucleus during electrophoresis.

The method of Singh et al., conducting the electrophoresis in alkaline conditions, allows both double and single strand breaks to be detected.

Solutions

• Lysing solution, pH 10: 2.5 M NaCl, 100 mM EDTA, 10 mM tris-(hydroxymethyl)-aminomethane and, added just before use: 1 % Triton-X and 10 % DMSO

• Electrophoresis buer, prepared freshly before each run: 1 mM EDTA (pH 10) and 300 mM NaOH

• Neutralisation buer, pH 7.5: 0.4 M tris-(hydroxymethyl)-aminomethane

• Phosphate buered saline (PBS), free of Mg²⁺ and Ca²⁺

• Normal melting point agarose (NMPA): 1 % w/v in PBS (e.g. 0.5 g in 50 mL)

• Low melting point agarose (LMPA): 0.5 % w/v in PBS

Sample preparation The process is summarised in Figure 8. Earthworm coelomocytes can be damaged by white light, therefore everything was performed in a room sparsely lit by yellow light.

Figure 8: Illustration depicting the steps in the sample preparation prior to scoring the comets in a microscope. See the text for details.

The initial step is to extract coelomic uid from the worm. This was done by direct extraction using a syringe, because if successful, this extraction method would be a time-saving method compared to the more time-consuming extrusion uid method, where worms are placed in an irritating solution and secrete cells as a response to this, whereupon the worm is removed and the solution additionally puried and diluted.

Rinsed worms were held so that the part just posterior of the clitellum was bent in a loop. The body wall of the worms were poked lightly with a syringe, causing them to excrete yellow coleomic uid. Approximately 20 µL of the excreted uid was drawn into the syringe, which already contained 20 µL of PBS. The PBS-coelomic uid mix, i.e. the cell suspension, was pipetted into an eppendorf tube on ice, and the cell concentration and viability was determined (see below). 10-20µL of the cell suspension was mixed with 60-70 µL of low melting point agarose (LMPA) held at 40 ^◦C. The LMPA-cell suspension mix was pipetted on to an already prepared microscope slide, coated with a layer of normal melting point agarose (NMPA), and a cover slip was placed on top. The LMPA

layer was allowed to coagulate on ice, after which the cover slip was removed. Another 75 µL of LMPA was pipetted on top of the coagulated LMPA layer, a cover slip was added and removed after coagulation. The positive controls were exposed to UV radiation for 2.5 minutes at 120 000 µJ in an Ultralum UVC-508 DNA crosslinker. Then, the slides were kept in coplin jars containing lysing solution for one hour.

Subsequently, the slides were carefully removed, washed twice with cold distilled water, and placed in an electrophoresis box containing alkaline electrophoresis buer. In order for DNA unwinding to occur, the slides were left in the alkaline buer for 30 minutes before electrophoresis start. The electrophoresis ran for 30 minutes at 25 V and 300 mA.

Subsequently, the slides were washed once with cold distilled water and ve times with neutralisation buer. The slides were drained and allowed to dry at room temperature.

They were kept in a microscope slide box until examined under a microscope.

Cell concentration and viability The viability of the coelomocytes was assessed by the Trypan Blue exclusion method, which is based on that cells with an intact plasma membrane, i.e. living cells, do not take up the Trypan Blue dye, whereas dead cells do, and therefore appear blue when examined under a microscope. 5 µL of Trypan Blue solution (0.4 %), 30µL of PBS (pH 7.3) and 20µL of cell suspension were mixed and left to react for 5 minutes, whereafter investigated on a Neubauer haemacytometer. 10 large square counts were done for each cell suspension, ve on each side of the haemacytometer (middle and corner squares). The concentration (cells/mL) of the cell suspension was determined by multiplying the average count squares by 10 000, whereas the viability was expressed as the average percentage of living cells. Preliminary trials were done, where cell suspensions from 10 worms taken directly from the batch cultures were assessed, to see wether the syringe extraction gave enough undamaged cells to score for DNA strand breaks.

Scoring of the comets Solutions

• TE buer: 10 mM Tris-(hydroxymethyl)-aminomethane and 1 mM EDTA, pH 8

• SYBR Gold dye solution: 200 µL of SYBR Gold nucleic acid gel stain diluted in 1,8 mL of DMSO

The scoring was done at the National Institute of Public Health in Oslo, Norway, using a Leica DMLB uorescence microscope with a camera, and the computer analysis program

Kinetic Imaging Komet 4.0. This image analysis program calculates a range of parame-ters, including the olive tail moment (OTM), tail extent moment (TEM), tail length and the distribution of DNA between the head and the tail of the comet. OTM and TEM were chosen as the parameters with which to present the results. OTM is dened as

T MOlive = (CGtail−CGhead)×DN Atail

whereCG_tail andCG_headis the center of gravity of the DNA in the comet's tail and head, respectively, and DN A_tail is the percentage of the total DNA of the comet located in the tail. TEM is dened as

T M_ext=L×DN A_tail

where L is the length of the tail and DN A_tail is as described above.

The cells were stained before scored. This was done by placing four slides in a plastic box containing 20 mL of TE-buer and 10 µL of SYBR Gold dye solution, and shaking the box very slowly on a microshaker for 15 minutes, in order for the stain to be distributed homogeneously. Subsequently, the slides were washed three times with distilled water.

Cover slips were placed on the slides, and they were examined under the microscope (40x/0.65 objective). Images of individual cells were taken with the camera, and Kinetic Imaging Komet 4.0 calculated various parameters based on the images. All these steps were performed in a dimly lit room. Ideally, 100 cells per slide were scored. However, the amount and quality of cells on the slides diered, so the amount of scored cells was adjusted accordingly.

Only the cells sampled after 168 hours of exposure were used when presenting the results, because this was the time group with the purest slides.