Cometassay

DU145 and PC3 cells were transfected with 20 nM of control or radiation sensitizing miRNAs and then treated with 4 Gy of IR 48 h post transfection. At 4 h after IR, the CometAssay® from Trevigen (Gaithersburg, MD, USA) was performed according to manufacturer instructions. Briefly, cells were mixed with low melting point agarose and plated on microscope slides and allowed to gel. Cells were then lysed under neutral buffer followed by rinse in TBE buffer (10.8% [w/v] tris base, 5.5% [w/v] boric acid, 0.93% [w/v] EDTA). After electrophoresis in TBE buffer for 40 min at 1 V/cm, slides were washed in water and dehydrated with ethanol, air dried overnight and then treated with SYBR Green for DNA staining. Comets were imaged by Imager.Z1 fluorescent microscopy (Carl Zeiss AG, Oberkochen, Germany) and analyzed using CometScore. The tail moment was calculated as the average of at least 50 comets.

H1299-IL24 cells (0.2 × 10⁶/well) seeded in six-well plates were treated with or without doxycycline (1 µg/mL). The cells were harvested at 48 h and 72 h after treatment and subjected to Comet assay (Trevigen, Gaithersburg, MD, USA) [59 (link)]. The Olive tail moment was determined by screening 10 cells per field for five fields (50 cells in total) in each sample.

Twenty four hours after monocytes (U937) being treated with JUUL pods and similar pod flavors, the cells were prepared for DNA damage assessment by Comet assay according to the manufacturer’s protocol (Trevigen, MD). Briefly, the alkaline procedure was followed and approximately 5000 cells per sample were used and stained with SYBR gold 1:10,000. EVOS imaging system was used to capture the fluorescence images. The images were inverted for the Opencomet analysis. The olive tail moment was obtained using Opencomet software for the representative images, and the percent change was calculated in comparison to the control group.

DNA Comet Assay was used to detect DNA-Double Strand Breaks following the instructions from Comet Assay® (Trevigen, MD USA) protocols.

U2OS cells were seeded in 6-well plates at a density of 1 × 10⁴ cells per well and treated with 500 μM DFMO for 72 h. Cells were washed with phosphate-buffered saline (PBS) twice and incubated with 125 nM olaparib for 24 h or irradiated with a 10 Gy ionizing radiation. Cells were then cultured with fresh medium containing 500 μM DFMO for the indicated recovery time. Comet assay (Trevigen) under alkaline condition was conducted according to the manufacturer’s instructions. Briefly, cells were mixed with low-melting temperature agarose and spread onto slides. Slides were immersed in lysis buffer and then in unwinding solution for 1 h at 4 °C. Slides were then subjected to electrophoresis at 21 volts for 1 h at 4 °C, followed by staining with SafeView Plus dye (Applied Biological Materials) at room temperature for 40 min. Fifty randomly selected cells per sample were captured using a Leica DM6000B upright microscope system. The tail moment was computed as the percentage of DNA in the comet tail multiplied by the tail length in the individual nuclei using the CometScore software (TriTek).