Sybr gold nucleic acid gels stain

Endogenous DNA damage levels in PBMCs were measured by single-cell gel electrophoresis (comet assay) under alkaline conditions, measuring single-strand breaks (SSBs) and/or double-strand breaks (DSBs) as previously described (15 (link)). Briefly, PBMCs (5x10⁴ cells) on comet slides were lysed and electrophoresis was performed for 30min at 1V/cm, 4°C. Slides were stained with SYBR Gold Nucleic Acid Gels Stain (Thermo Fischer Scientific, #S11494) and visualized using fluorescence microscope (Zeiss Axiophot). Comet images were analyzed by the Open Comet in ImageJ software. Olive tail moment (OTM) of at least 200 cells/treatment condition was evaluated in order to quantify DNA damage.

Alkaline or a neutral comet assay was performed as described previously [13 (link)]. That is, PBMCs (5 × 10⁴ cells) were suspended in 1% low melting point (LMP) agarose in phosphate-buffered saline (PBS) at 37 °C and spread on comet slides (CometAssay^® HT Slide, Trevigen, MD, USA). After gentle cell lysis (Trevigen; #4250-050-01) and denaturation of the DNA, electrophoresis was carried out at 21 V, 300 mA, and 30 min at 4 °C. Thereafter, comet slides were washed in distilled water, fixed in 70% ethanol, stained with SYBR Gold Nucleic Acid Gels Stain (Thermo Fischer Scientific, Waltham, MA, USA, #S11494) and analyzed by fluorescence microscope (Zeiss Axiophot, Carl Zeiss QEC GmbH, Germany). Comet images were analyzed with ImageJ Analysis/Open Comet v1.3.1, an open-source software tool providing automated analysis of comet assay images.