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Reference cells and ploidy in the comet assay.

Brunborg G, Collins A, Graupner A, Gutzkow KB, Olsen AK - Front Genet (2015)

Bottom Line: The analysis of testicular cell suspensions provides information on cell type specific composition, susceptibility to genotoxicants, and DNA repair.They can also be used - in combination with a reference curve - to quantify the DNA lesions induced by a certain treatment.Fish cells of a range of genome sizes, both greater and smaller than human, are suitable for this purpose, and they are inexpensive.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemicals and Radiation, Division of Environmental Medicine, Norwegian Institute of Public Health, Oslo Norway.

ABSTRACT
In the comet assay single cells are analyzed with respect to their level of DNA damage. Discrimination of the individual cell or cell type based on DNA content, with concomitant scoring of the DNA damage, is useful since this may allow analysis of mixtures of cells. Different cells can then be characterized based on their ploidy, cell cycle stage, or genome size. We here describe two applications of such a cell type-specific comet assay: (i) Testicular cell suspensions, analyzed on the basis of their ploidy during spermatogenesis; and (ii) reference cells in the form of fish erythrocytes which can be included as internal standards to correct for inter-assay variations. With standard fluorochromes used in the comet assay, the total staining signal from each cell - whether damaged or undamaged - was found to be associated with the cell's DNA content. Analysis of the fluorescence intensity of single cells is straightforward since these data are available in scoring systems based on image analysis. The analysis of testicular cell suspensions provides information on cell type specific composition, susceptibility to genotoxicants, and DNA repair. Internal reference cells, either untreated or carrying defined numbers of lesions induced by ionizing radiation, are useful for investigation of experimental factors that can cause variation in comet assay results, and for routine inclusion in experiments to facilitate standardization of methods, and comparison of comet assay data obtained in different experiments or in different laboratories. They can also be used - in combination with a reference curve - to quantify the DNA lesions induced by a certain treatment. Fish cells of a range of genome sizes, both greater and smaller than human, are suitable for this purpose, and they are inexpensive.

No MeSH data available.


Related in: MedlinePlus

Fish erythrocytes and human PBMN cells exposed to ionizing radiation. Left y-axis, medium level of DNA damage (Tail %DNA) vs. X-ray dose (x-axis). Right y-axis, median Total Fluorescence Insensity (arbitrary unit) vs. X-ray dose. Data are derived from Figure 3.
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Figure 4: Fish erythrocytes and human PBMN cells exposed to ionizing radiation. Left y-axis, medium level of DNA damage (Tail %DNA) vs. X-ray dose (x-axis). Right y-axis, median Total Fluorescence Insensity (arbitrary unit) vs. X-ray dose. Data are derived from Figure 3.

Mentions: The mean TFI for turbot FE in Figure 3A is approximately 3–4 times lower than for HPBL, as expected from the genome size differences (see also Figure 4). It is apparent from the figure that there are no cells with TFI between 70,000 and 100,000. The absolute threshold value is subject to inter-experimental variations depending on the intensity of the lamp and the staining of DNA. Traditional fluorescent light sources (Mercury Xenon) produce less light with time of use, but newer technologies (liquid light guide combined with metal halide or LED light) solve this latter problem.


Reference cells and ploidy in the comet assay.

Brunborg G, Collins A, Graupner A, Gutzkow KB, Olsen AK - Front Genet (2015)

Fish erythrocytes and human PBMN cells exposed to ionizing radiation. Left y-axis, medium level of DNA damage (Tail %DNA) vs. X-ray dose (x-axis). Right y-axis, median Total Fluorescence Insensity (arbitrary unit) vs. X-ray dose. Data are derived from Figure 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4343028&req=5

Figure 4: Fish erythrocytes and human PBMN cells exposed to ionizing radiation. Left y-axis, medium level of DNA damage (Tail %DNA) vs. X-ray dose (x-axis). Right y-axis, median Total Fluorescence Insensity (arbitrary unit) vs. X-ray dose. Data are derived from Figure 3.
Mentions: The mean TFI for turbot FE in Figure 3A is approximately 3–4 times lower than for HPBL, as expected from the genome size differences (see also Figure 4). It is apparent from the figure that there are no cells with TFI between 70,000 and 100,000. The absolute threshold value is subject to inter-experimental variations depending on the intensity of the lamp and the staining of DNA. Traditional fluorescent light sources (Mercury Xenon) produce less light with time of use, but newer technologies (liquid light guide combined with metal halide or LED light) solve this latter problem.

Bottom Line: The analysis of testicular cell suspensions provides information on cell type specific composition, susceptibility to genotoxicants, and DNA repair.They can also be used - in combination with a reference curve - to quantify the DNA lesions induced by a certain treatment.Fish cells of a range of genome sizes, both greater and smaller than human, are suitable for this purpose, and they are inexpensive.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemicals and Radiation, Division of Environmental Medicine, Norwegian Institute of Public Health, Oslo Norway.

ABSTRACT
In the comet assay single cells are analyzed with respect to their level of DNA damage. Discrimination of the individual cell or cell type based on DNA content, with concomitant scoring of the DNA damage, is useful since this may allow analysis of mixtures of cells. Different cells can then be characterized based on their ploidy, cell cycle stage, or genome size. We here describe two applications of such a cell type-specific comet assay: (i) Testicular cell suspensions, analyzed on the basis of their ploidy during spermatogenesis; and (ii) reference cells in the form of fish erythrocytes which can be included as internal standards to correct for inter-assay variations. With standard fluorochromes used in the comet assay, the total staining signal from each cell - whether damaged or undamaged - was found to be associated with the cell's DNA content. Analysis of the fluorescence intensity of single cells is straightforward since these data are available in scoring systems based on image analysis. The analysis of testicular cell suspensions provides information on cell type specific composition, susceptibility to genotoxicants, and DNA repair. Internal reference cells, either untreated or carrying defined numbers of lesions induced by ionizing radiation, are useful for investigation of experimental factors that can cause variation in comet assay results, and for routine inclusion in experiments to facilitate standardization of methods, and comparison of comet assay data obtained in different experiments or in different laboratories. They can also be used - in combination with a reference curve - to quantify the DNA lesions induced by a certain treatment. Fish cells of a range of genome sizes, both greater and smaller than human, are suitable for this purpose, and they are inexpensive.

No MeSH data available.


Related in: MedlinePlus