Sybr green 1 dye

DNA DSBs in HT22 cells were evaluated using the CometAssay® Kit (Trevigen Inc., MD, USA) under neutral condition. According to the manufacturer’s instruction, cells were collected and resuspended in ice cold PBS (Ca²⁺ and Mg²⁺ free) to a concentration of 1 × 10⁵ cells/ml. After mixing 50 μl of cell suspension into 500 μl of molten LMAgarose (1% low-melting agarose), 50 μl of the mixture was immediately taken and evenly spread onto a comet slide. The slide was then incubated at 4 °C in the dark for 30 min, then transferred to prechilled lysis solution for overnight at 4 °C. Electrophoresis was performed in Neutral Electrophoresis Buffer at 21 V for 50 min at 4 °C the next day. After electrophoresis, slides were sequentially immersed in DNA Precipitation Solution and 70% ethanol for 30 min each at RT. At last, slides were dried and stained with diluted SYBR® Gold for 30 min, followed by air dry at 37 °C and staining with diluted SYBR Green I dye (Trevigen, 1:10 000 in Tris–EDTA buffer, pH 7.5) for 30 min. Images were taken under a confocal scanning laser fluorescent microscope (FV1200, Olympus, Japan) and analyzed using Comet Score15 Software.

The comet assay (Trevigen, Gaithersburg, MD) was performed according to manufacturer's protocol using neutral conditions. After lysis, the slides were washed twice with 1× Tris-borate EDTA buffer solution, pH 8.3 (TBE) for 10 min. The slides were placed in a horizontal electrophoresis chamber and covered with TBE buffer. Electrophoresis was carried out at the rate of 1.0 V/cm for 20 min. The slides were removed from the electrophoresis chamber, washed in deionized water for 5 min and immersed in ice cold 100% ethanol for 5 min. Subsequently, the slides were air dried, DNA was stained with 50 μl of SYBR Green I dye (Trevigen, 1:10,000 in Tris-EDTA buffer, pH 7.5) for 20 min in the refrigerator and immediately analyzed using upright fluorescence microscope (Nikon, Melville, NY), and data was analyzed using CometScore (TriTek, Sumerduck, VA).

A549/CR and H460/CR cells treated with PGG (12.5 and 25 μM) for 48 h were evaluated for DNA damage using a Trevigen Comet Assay™ kit (Trevigen Inc., Gaithersburg, MD, USA). The cells were subjected to regular Comet assay, and the isolated DNAs were stained with SYBR Green I dye (Trevigen, 1:10,000 in Tris–EDTA buffer, pH 7.5) for 20 min and visualized using an Axio vision 4.0 fluorescence microscope (Carl Zeiss Inc., Dublin, CA, USA).