Comet assay kit

Alkaline comet assay was conducted using a comet assay kit (Biotechne, #4250-050-K, Minneapolis, MN, USA) following the manufacturer’s instructions. DNA was stained with Hoechst 33342 (Thermo Scientific, #62249) and fluorescence images were captured using a fluorescence microscope at × 400 magnification.

Comet Assay was performed using a CometAssay Kit (Cat#4250-050-K, Bio-Techne, Minneapolis, MN, USA), according to manufacturer’s instructions. Briefly, cells were seeded into 6-well plates and treated next day with 0.1 µM of doxorubicin for 30 min. After media change, cells were cultured for 2 or 24 h and then combined with molten LMAgarose (Cat#4250-0505-02, Bio-Techne) at a ratio of 1:10 and immediately spread onto both wells of a CometSlide (Cat#4250-050-03, Bio-Techne). Upon lysis with Lysis Solution (Cat#4250-050-01, Bio-Techne) and immersion in Alkaline Unwinding Solution (200 mM NaOH, 1 mM EDTA, pH > 13), slides were then placed into the electrophoresis slide tray of the CometAssay ES Unit (Cat#4250-050-ES, Bio-Techne) covered with Alkaline Electrophoresis Solution (200 mM NaOH, 1 mM EDTA, pH > 13). After electrophoresis, slides were washed, dried, stained with SYBR Gold (Cat# S11494, Thermo Fisher Scientific, Waltham, MA, USA) and scanned using 10× objective with the Cytation 3 Cell Imaging Multi-Mode Reader (Cat#CYT3MV, Agilent). Each well was scanned using a 10 × 10 grid (100 images total), images processed and analysed using CometScore 2.0 to calculate % of DNA in tail and Tail length (µm).

Endogenous level of DNA damage in HEK293 cells was evaluated using the CometAssay kit (Trevigen) according to the manufacturer’s instructions. Control cells were treated with 1 mM MMS (Sigma) for 1 h to induce DNA damage. Cells were harvested by trypsinization and 500 cells were mixed with LMAgarose and spread on the slide. Slides were dried 20 min in 4 °C, incubated 40 min in kit lysis solution in 4 °C and 20 min in unwinding solution (300 mM NaOH, 1 mM EDTA, pH 13) in room temperature. Electrophoresis was performed in the unwinding solution in 4 °C, 300 mA, 30 min. Slides were washed twice in water, once in 70% EtOH, dried 15 min in 37 °C and stained with SYBR Gold (Thermo) for 30 min. Images were taken using Zeiss Axio Imager MI with 20 x objective.

A comet assay was performed by using a CometAssay kit (Trevigen). Briefly, spermatozoa (1 × 10⁵ cells/mL) in PBS were mixed with melted agarose and then immediately pipetted on to a CometSlide. The slides were immersed for 1 h on ice in pre-chilled lysis buffer, incubated for 18 h in lysis buffer containing 500 μg/mL proteinase K60 (link). and then subjected to electrophoresis. Finally, the slides were immersed in chilled 70% ethanol and was stained with SYBR Green. The results were expressed as the ‘tail length’ and ‘% DNA in tail’, using TriTek Comet Score-Freeware v1.5 (TriTek Corp., Sumerduck, VA, USA).

DNA damage was detected by single-cell gel electrophoresis by using the Comet assay kit (Trevigen) [44 (link)]. MDA-MB-231 cells were added to a culture dish containing the medium. After treatment with different concentrations of the complexes (0, 5, and 10 μM) at 37 °C for 72 h, the cells were collected and washed with PBS three times. A solution of the cell suspension and melted low-melting-point agarose gel was mixed at a volume ratio of 1:7, and the equilateral sample was immediately transferred to a Comet Slide TM slide. The slides were washed twice with PBS, placed into an electrophoresis tank containing comet electrophoresis solution, and electrophoresed at 25 V for 30 min. After electrophoresis, the slides were washed twice with PBS. Finally, the cells were stained with EB and observed under a fluorescence microscope. Cells were randomly selected from each slide to take photos, and the tail moment was analyzed.