Protocol for assessing DNA damage levels in vivo in rodent testicular germ cells in the Alkaline Comet Assay
Few protocols are available for retrieving male germ cell-specific information on DNA damage dynamics or assessing the potential harmful effects of environmental contaminants, drugs, or lifestyle factors related to genotoxicity. Here, we present a protocol for evaluating testicular germ cell genotoxicity using a modified version of the alkaline comet assay in rodent testicular germ cells. The protocol includes experimental design, preparation of testicular cell suspensions, comet analysis, germ cell-specific scoring, and data curation methods to collect information on testicular male germ cells, specifically spermatids and primary spermatocytes. For complete details on the use of this protocol please refer to Olsen et al., in preparation HighlightsO_LIDNA damage levels can be specifically measured in testicular germ cells using the developed revised version of the alkaline comet assay C_LIO_LI1C spermatids as well as 4C primary spermatocytes can be assessed C_LIO_LIBoth manual- and modeling-based approaches were developed that facilitate user-friendly protocols to select 1C spermatid comets. C_LIO_LIThis protocol expands the limited methodologies available to study germ cell DNA damage dynamics C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/624648v7_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1c1e54eorg.highwire.dtl.DTLVardef@b569e9org.highwire.dtl.DTLVardef@3c74adorg.highwire.dtl.DTLVardef@110122e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG