Quantifying DNA Damage with Alkaline Comet Assay

Alkaline comet assays were performed using the R&D Systems CometAssay single-cell gel electrophoresis assay as described by the manufacturer. Briefly, individual cells are embedded in a thin agarose gel on a microscope slide. All cellular proteins are then extracted from the cells by lysing. The DNA is allowed to unwind under alkaline/neutral conditions. Following the unwinding, the cell/DNA is subjected to electrophoresis, allowing the broken DNA fragments or damaged DNA to migrate away from the nucleus. After staining with a DNA-specific fluorescent dye such as ethidium bromide or propidium iodide, the “cell/DNA” is imaged, and the amount of fluorescence in head and tail and length of tail was determined. The image obtained resembles a “comet” with a distinct head and tail. The head is composed of intact DNA, while the tail consists of damaged (single-strand breaks or DSBs) or broken pieces of DNA. The extent of DNA liberated from the head into the tail of the comet is directly proportional to the amount of DNA damage.
Various cell types were evaluated in comet assays. 3T3 cells were treated with ICA or vehicle for 24 hours, irradiated at the doses indicated, and trypsinized at either 5 or 20 min. After irradiation, cells were washed once in ice-cold 1× PBS (Ca²⁺ and Mg⁺⁺ free) and suspended in ice-cold 1× PBS (Ca⁺⁺ and Mg⁺⁺ free) at 1 × 10⁵ cells/ml. Cells were mounted on comet slides, electrophoresed, and stained with Sybr Gold, according to manufacturer specifications. Slides were imaged by epifluorescence microscopy, and images were collected of all detectable comets on each slide. For splenocytes and oocytes, isolation was performed as described as above, X-irradiated in RPMI and M2 media, respectively, and the comet assay was performed as above. In experiments where mice were treated with vehicle or ICA, the medium did not contain ICA or vehicle after cells were isolated from the animal, during radiation ex vivo, or after radiation. However, for experiments with untreated animals, cells were treated with ICA/vehicle ex vivo, and the medium contained ICA or vehicle in all postisolation manipulations. For 3T3 cells and splenocytes, images were analyzed using Open Comet software (https://cometbio.org/) to determine percent tail DNA, olive moment, and tail moment. Because of the difference in cell size, oocytes were not easily recognized by Open Comet; thus, manual measurements were obtained using Intelligent Imaging Innovations Slidebook software (V6.1). For each oocyte comet, masks were used to calculate the area and intensity of the head of the comet alone, as well as that of the whole comet (head + tail). Percent tail DNA was calculated as follows: (i) head area × head intensity = head DNA; (ii) total comet area × total intensity = total comet DNA; (iii) total comet DNA − head DNA = tail DNA; and (iv) (tail DNA/total comet DNA) × 100 = % tail DNA.