Alkaline cometassay kit

DNA single- and double-stranded breaks were determined by the Alkaline CometAssay kit (Trevigen) according to the manufacturer’s protocol. Briefly, after treatment with the TOP1 inhibitors, cells were harvested, washed in 1xPBS, and combined at 3x10⁵cells/ml with molten LMAagrose in a 1:10 (v/v) ratio. Fifty μl of the combined mixture was added onto the comet slide. After the gel was solidified at 4°C, slides were immersed in 4°C lysis solution for 30 min and subsequently incubated in alkaline unwinding solution for 20 min at RT in the dark. Alkaline electrophoresis was carried out at 1 V/cm and 300 mA for 40 min at 4°C. Slides were rinsed twice in deionized H2O for 5 min each, then in 70% ethanol for 5 min and air-dried overnight. DNA was stained with 100 μl SYBR Gold for 30 min, briefly rinsed in water, and allowed to air-dry. Fluorescent signals were visualized using fluorescence microscopy and quantified by using ImageJ plugin OpenComet (version 1.3)

The alkaline comet assay is used for the detection of DNA strand breaks in cells or nuclei following exposure to potentially genotoxic materials [22 ]. Under alkaline conditions (>pH 13), the comet assay can detect single and double stranded breaks, resulting, for example, from direct interactions with DNA, alkali labile sites or as a consequence of transient DNA strand breaks resulting from DNA excision repair [22 ]. Cells at approximately 60% confluence in 6-well dishes containing 1 ml of RPMI serum free media were exposed in triplicate, removed from the plate by scraping, and the Trevigen Alkaline Comet Assay kit was used to conduct the comet assay. Comet images were captured at 4× magnification on a microscope equipped with epifluorescence and a CCD camera. All slides for analysis were randomly coded and scored “blinded” to prevent scoring bias. At least 150 scoreable comets without overlapping tails and not located at the edge of the slides per treatment per cell line were analyzed using Trevigen Comet Analysis Software. The percent tail DNA (also known as percent tail intensity) was used for the evaluation and interpretation of results, and was determined by the DNA fragment intensity in the tail expressed as a percentage of the cell's total intensity.