Owl easycast b1a

Neutral comet assays were performed according to the Trevigen protocol. Cells were trypsinized and resuspended in PBS at a density of 3 × 10⁵ cells/mL. Cells were mixed at a 1:10 v:v ratio with molten 1% low melting point agarose (Trevigen) at 37°C, and 50 µL of cell-agarose solution was dropped onto Flare slides (Trevigen). The agarose was allowed to solidify at 4°C, and slides were soaked in ice-cold Comet lysis buffer (Trevigen) and incubated at 4°C overnight. Slides were then incubated for 30 min at 4°C in cold neutral electrophoresis buffer (100 mM Tris, 300 mM sodium acetate at pH 9.0). Slides were carefully immersed in neutral electrophoresis buffer in a Thermo Scientific Owl EasyCast B1A mini gel electrophoresis system gel tank and electrophoresis was carried out at 15 V (1 V/cm) for 1 h at 4°C. Slides were sequentially incubated for 30 min in DNA precipitation solution (1 M ammonium sulfate, 95% ethanol) and 70% EtOH. Slides were dried and 200 µL of SYBR Gold DNA staining solution (1:10,000 SYBR Gold, 10 mM Tris at pH 7.5, 1 mM EDTA) was dropped onto each sample. Slides were incubated 30 min at room temperature, washed twice with H₂O, and completely dried before mounting with ProLong Gold. Images were captured using NIS elements software with a Nikon i90 microscope and tail moments were analyzed with the OpenComet software integrated in Fiji (Gyori et al. 2014 (link)).