The comet assay, also known as the single-cell gel electrophoresis assay, is a method used to detect D N A damage in almost all nucleated cells. The comet assay has significant advantages over other genotoxicity detection assays, but it can lead to highly variable results due to its sensitivity to subtle changes in the experiment. The purpose of this chapter is to provide background information and detailed standardized procedures related to the alkaline comet assay in environmental toxicity testing. We will describe the pitfalls associated with the comet assay and the considerations of the procedure, as well as briefly discuss ways to improve the common alkaline comet assay so that it can be better applied to a wide range of situations in the study of environmental toxicology.
By Martin, this experiment is from "Environmental Genomics Experiment Guide".
Operation method
Experiments on the application of the comet assay to environmental toxicology Move I. Materials 1. Solution 1.2 Glassware and laboratory equipment (1) Pipette. (2) Pipette tips (10, 20, 100, 200, 1000 uL). (3) Graduated flasks (100 m L, 500 m L and I L). (4) Measuring cylinders (100 m L, 500 m L and 1L). (5) Beakers (250 m L, 500 m L and 1L). (6) Petri dishes (120 mM diameter). (7) Wash bottle (for ultrapure water). (8) Ice box. (9) Freezing tube (1.0 m L ) (10) Eppendorf tube (0-5 m L, I.5 m L). (11) Centrifuge tube with screw cap (50 m L ). (12) Plastic/glass disk (e.g., lid or sump of pipette container). (13) Microscope slides. (14) Microscope cover slips. 1.3 Instruments (1) p H meter. (2) Analytical balance. (3) Magnetic stirrer and stirring bar. (4) Power supply to provide low voltage and high current (e.g., 300 V , 2000 m A ). (5) Horizontal electrophoresis apparatus. (6) Deterrent. (7) Scissors. (8) Gelbond glue (agarose gel support medium) (Mandel cat. no. 53740 G B 1638). (9) L a b T e k I [small chamber (NalgeNunccat. no. 154461-B ). (10) Heater or water bath. (11) Computer with Comet image analysis software (Kinetic Komet 5. 5 or other). (12) Burst light microscope with 40x oil lens (Zeiss AxioPlan U or other) and suitable excitation/emission filters (e.g. S Y B R Gold: 300 n m excitation light, 495/537 n m emission light, stained nucleic acids) Calibration of the comet assay can be performed by evaluating the cellular DNA damage induced by ionizing radiation (X-rays or γ-rays) or chemical treatment (hydrogen peroxide, cyclophosphamide, or MMS) (see Note 7) (I, 4, 5). A dose-effect curve was made to determine the sensitivity of this technique (Fig. 2). Calibration is required each time a new species and a different cell line is used. Some laboratories ensure the consistency of each comet assay by performing a positive control (e.g., radiation or chemical treatment) at the same time Apoptotic or necrotic cells do not exhibit the typical comet image. Instead, their heads are small or absent while the comet tails are large and diffuse (Fig. 3 ) These cells are often referred to as teardrop cells, ghost cells, or hedgehog cells (1, 4). Such cells can be induced to arise by cytotoxic and/or nongenetic toxicants and should be excluded from analysis. Cells exposed to genetic toxicants can also show this type of image, at which point they should then be included in the data analysis. Therefore, concurrent cytotoxicity measurements of cell suspensions are required to determine the cause of severe cell damage and whether these cells should be included in the data analysis. The comet assay can be performed on almost all nucleated cells, provided they are alive. However, in some genera (e.g., birds), whole blood is not suitable for comet assays because more than 80% of the cells appear to be "ghost cells" or "hedgehog cells," possibly due to degradation and functionally inert DNA/RNA in nucleated mature erythrocytes. In this case, leukocytes need to be separated from nucleated erythrocytes for comet assays. This phenomenon is not present in whole blood cells of amphibians. To quantify the D N A damage, the migration of D N A was observed under a fluorescence microscope after gel staining (e.g., E B, S Y B R Gold, S Y B R G r e e n ), and the comets were scored by suitable software packages such as K o m e t 5. 5 (Kinetic Imaging, Nottingh a m , U K ), C o m e t Assay F (Perceptive Instruments, Suffolk, U K ) to score comets. Damaged cells will show an image similar to that of a comet in the starry sky, with a long D N A-containing tail migrating out of the center of the nucleus of the damaged cell. Three important values are applied to the degree of damage: the comet tail length, the tail torus (the product of the comet tail length and the percentage of D N A in the comet tail) or the Oliv e tail distance (the distance from the center of gravity of the comet tail to the center of the comet's head multiplied by the percentage of D N A in the comet tail), and the percentage content of D N A in the tail (7, 8). Since different image analysis software calculates these metrics in different ways, the jury is still out on which metric is most effective. When the Comet assay cannot be performed immediately after sample collection, samples can be frozen in liquid nitrogen until a suitable period of time, provided that they are placed in a suitable cellular freezing solution (11-13). We have found that either magnesium-free PBS with 10% DMSO or magnesium-free PBS with 10% DMSO and 20 mMol ZL EDTA (12) can be used as a suitable cell freezing solution for blood samples. Samples should be thawed in a water bath at room temperature and immediately followed up. However, data from comet experiments performed on thawed samples cannot be directly compared with data from non-thawed samples because the thawing process raises the background value of DNA migration. Therefore, the control experiments should also be controlled under the same conditions (cold freezing). Three modifications of the common alkaline comet assay can also be powerful tools for studying environmental toxicology and genomics. First, the comet assay can be combined with fluorescence in situ hybridization (F IS H ) to determine in which region of a gene a D N A break occurs (14, 15). The second method, the comet-DNA dispersion assay, distinguishes between types of cell death [e.g., apoptosis (programmed cell death)] and necrosis (cells that die in injury or disease) by precipitating D N A with ethanol without electrophoresis in the comet assay (16). In addition, DNA duplex breaks can also be detected by performing a comet assay in neutral rather than alkaline buffer (5, 17). This technique also allows evaluation of D NA damage in germ cells, which typically have high levels of alkaline destabilizing sites (5, 18, 19) (see Note 6). Caveat Inhabiting whole blood cells need to be stained for about 30 min. For more product details, please visit Aladdin Scientific website.
One of two methods is preferred for assaying cellular activity. The first is with the double staining activity assay (6). In this assay, equal volumes of cell suspension and E B / synaptophysin diacetate working solution (see 2 . 7 in 1 ) are mixed and then dropped on either side of a blood cell counting plate (10 fxL). Both dead and living cells will be counted simultaneously by fluorescence microscopy . Metabolically competent cells (living cells) convert fluorescein diacetate to metabolic incendiary light by cytosolic esterases
Metabolically competent cells (living cells) show green fluorescence through the conversion of fluorescein diacetate into metabolic incendiary fluorescein by cytosolic esterase, while metabolically incompetent cells (dead cells) show red coloration due to the altered permeability of the cell membrane, which is stained by ethidium bromide. Another method is the Tapan blue exclusion assay. In this assay, equal volumes of cell suspension and Tapan Blue solution are mixed and placed on both sides of a blood cell counting plate (IO mL), and both dead and living cells are counted through a normal microscope over a certain period of time (usually within 2 to 5 m i n ). Colored cells are not
The colorless cells are inactive, while the colorless and transparent cells are living cells. In general, samples with a proportion of viable cells below 70% to 75% of the negative control should not be analyzed subsequently. 
When exposed to low doses of genotoxic substances, a rapid increase in tail length ensues, but at higher concentrations the change in tail length reaches a plateau (9). However, the amount of D N A in the comet tail region is able to increase continuously with increasing toxicant dose, theoretically from 〇 ~ 100% (7). Therefore, the comet tail density continues to increase with increasing dose rather than tail length (Fig. 2). Since tail moments are calculated based on length, it has been suggested that comet tail density or comet tail D N A percentage should be used as the best measure of genotoxicity.
7. Other comet experimental techniques relevant to environmental toxicology