Reference cells and ploidy in the comet assay

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.

Technical recommendations to perform the alkaline standard and enzyme-modified comet assay in human biomonitoring studies

“…Even better, reference cells may be included in the same samples if they can be distinguished from (e.g.) PBMCs during scoring; such methods have recently been described based on the use of blood cells from fish with DNA content much lower than that in humans [65]. Assay controls or internal reference cells will guarantee that intra-laboratory variability is properly…”

Controlling variation in the comet assay

“…When scoring, appropriate (different) filters are used to identify sample and standard cells, and this makes the process of scoring more laborious. A second approach uses as standards cells with a markedly different genome size compared with human—for example, erythrocytes of certain fish species (Brunborg et al, 2014 ). After scoring all the comets in the gel, they are sorted into two sets according to total comet fluorescence, which is proportional to genome size.…”

The comet assay: past, present, and future

“…A further application of the comet assay is as a valuable experimental tool for human biomonitoring as well as in clinical studies. Collecting blood or tissues is not always feasible in all human subjects, and other sources of cells that can be collected non-invasively have been tested with the comet assay; for example, various types of epithelial cells (Rojas et al, 2014 ) as well as sperm (Cortés-Gutiérrez et al, 2014 ; Brunborg et al, 2015 ).…”

“…Internal standards—i.e., standard cells in the same gel as sample cells—would be ideal; but it is of course essential to be able to distinguish the two types of cell. Fish cells that are either larger or smaller in genome size compared to human cells have successfully been adopted for this purpose (Brunborg et al, 2015 ). These reference cells can be used in combination with a standard or calibration curve (established with cells given different doses of ionizing radiation), enabling a more precise quantification of DNA lesions expressed as a DNA break frequency rather than % tail DNA.…”