ASTM E2186-02a(2016)

5.1 A common result of cellular stress is an increase in DNA damage. DNA damage may be manifest in the form of base alterations, adduct formation, strand breaks, and cross linkages 5.1.1 The Comet assay can be easily utilized for collecting data on DNA strand breakage (9, 25, 26). It is a simple, rapid, and sensitive method that allows the comparison of DNA strand damage in different cell populations. As presented in this guide, the assay facilitates the detection of DNA single strand breaks and alkaline labile sites in individual cells, and can determine their abundance relative to control or reference cells (9, 16, 26). The assay offers a number of advantages; damage to the DNA in individual cells is measured, only extremely small numbers of cells need to be sampled to perform the assay (<10 000), the assay can be performed on practically any eukaryotic cell type, and it has been shown in comparative studies to be a very sensitive method for detecting DNA damage (2, 27) .

5.1.2 These are general guidelines. There are numerous procedural variants of this assay. The variation used is dependent upon the type of cells being examined, the types of DNA damage of interest, and the imaging and analysis capabilities of the lab conducting the assay. To visualize the DNA, it is stained with a fluorescent dye, or for light microscope analysis the DNA can be silver stained 5.2 A sufficient knowledge of the biology of cells examined using this assay should be attained to understand factors affecting DNA strand breakage and the distribution of this damage within sampled cell populations. This includes, but is not limited to, influences such as cell type heterogeneity, cell cycle, cell turnover frequency, culture or growth conditions, and other factors that may influence levels of DNA strand damage. Different cell types may have vastly different background levels of DNA single-strand breaks due to variations in excision repair activity, metabolic activity, anti-oxidant concentrations, or other factors. It is recommended that cells representing those to be studied using the SCG/Comet assay be examined under the light or fluorescent microscope using stains capable of differentially staining different cell types. Morphological differences, staining characteristics, and frequencies of the different cell types should be noted and compared to SCG/Comet damage profiles to identify any possible cell type specific differences. In most cases, the use of homogenous cell populations reduces inter-cell variability of SCG/Comet values. The procedures for this assay, using cells from many different species and cell types, have been published previously 5.3 The experimental design should incorporate appropriate controls, reference samples, and replicates to delineate the influence of the major sources of experimental variability.

1.1 This guide covers the recommended criteria for performing a single-cell gel electrophoresis assay (SCG) or Comet assay for the measurement of DNA single-strand breaks in eukaryotic cells. The Comet assay is a very sensitive method for detecting strand breaks in the DNA of individual cells. The majority of studies utilizing the Comet assay have focused on medical applications and have therefore examined DNA damage in mammalian cells in vitro and in vivo (1-4).2 There is increasing interest in applying this assay to DNA damage in freshwater and marine organisms to explore the environmental implications of DNA damage.