Horizontal electrophoresis chamber

The protocol of Collins (Collins et al. 2023 (link)) was applied with minor modifications. Cell suspensions were mixed with 0.7% low-melting-point agarose (LMPA) for the preparation of gels. After solidification, the embedded cells were lysed at 4 °C overnight (lysis solution: 2.5 M NaCl, 100 mM EDTA-Na₂, 10 mM Tris base, pH 10 and 1% Triton X-100 added just before use). Slides were then placed in alkaline electrophoresis solution (0.3 M NaOH, 1 mM EDTA-Na₂, pH > 12) for 20 min at 4 °C for unwinding and then electrophoresed in the same solution for 20 min at a voltage gradient of 0.8 V/cm across the platform in a horizontal electrophoresis chamber (Bio-Rad, Richmond, CA, USA). Finally, the slides were rinsed once with cold PBS (1 × , pH = 7.4), twice washed in cold distilled water and left to dry. After drying, slides were stained with 1 µM SYBR™ Gold for 30 min in the dark and then rinsed twice in distilled water.
The semi-automated image analysis system (Comet Assay IV; Perceptive Instruments) was used to evaluate 50 comets per gel. The percentage of DNA in the tail (% tDNA) was the descriptor used, and the median value of % tDNA from 100 comets was used to measure DNA damage for each condition.

Comet assays were performed using the OxiSelect Comet Assay Slides according to the manufacturer’s instructions (Cell Biolabs Inc., San Diego, CA). A cellular suspension in PBS (1 × 10⁵ cells per mL) was mixed 1:10 with molten low-melting-point agarose at 37 °C. Seventy-five microliters of this suspension were then placed within each well on the specially prepared microscope slide provided with the kit. Slides were incubated at 4 °C for 15 min to allow the agarose to solidify and then were placed in lysis buffer and incubated for 30 min at 4 °C in the dark. Denaturation was achieved by transferring the slides to a pre-chilled alkaline electrophoresis buffer (300 mM sodium hydroxide, 1.0 mM EDTA) and incubating for 30 min at 4 °C in the dark. The slides were then transferred into a horizontal electrophoresis chamber (BioRad Inc., Hercules, CA) and subjected to electrophoresis at 1 V/cm (30 V, 300 mA) for 15 min. Following electrophoresis, slides were washed 3 times with water, once with 70% ethanol, and air dried. The dried slides were stained with 100 μL of diluted Vista Green DNA dye (Cell Biolabs Inc., San Diego, CA), and cellular DNA was visualized using a FITC filter-fitted microscope. Comet images were scored visually on a scale of 0 to 4 as described by Collins [23 (link)], with 0 representing no damage and 4 representing severe damage.

PCR products were extracted with a mixture of chloroform and isoamyl alcohol (24:1). The DNA was dissolved in 9 μL of buffer (10 mM Tris (pH 9.0), 50 mM sodium chloride), 1 μL of 10 × dye (50 mM Tris (pH 8.27), 0.25% bromophenol blue, 60% glycerol) and applied to a horizontally positioned agarose gel (2% agarose, 1 × TAE, 0.6 μg/ml ethidium bromide). Electrophoresis was performed in 1 × TAE buffer for 35 min in a horizontal electrophoresis chamber (BioRad, Hercules, CA, USA) at 5 V/cm (Elf-8 power source, DNA-Technology, Moscow, Russia).