Sybr gold nucleic acid gel staining

An alkaline, single-cell gel electrophoresis assay developed in-house was used to quantify the DNA damage induced by PCP and the ZnO ENM exposure according to a protocol described by Watson et al. (Watson et al. 2014 ). Briefly, 10⁵ cells per well were loaded for each cell type in 96-well plates and were left to settle over 15 or 30 min for TK6 or Calu-3 cells, respectively. After excess cells were aspirated, the chips were rinsed once with PBS. Molten, low-melting point agarose was then overlaid onto the chip and allowed to set for 2 min at 4 °C. Following this step, the chip was submerged in ice-cold alkaline lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 9.5 with 0.5% Triton X-100). After overnight incubation, alkaline buffer (0.3 M sodium hydroxide and 1 mM Na2EDTA in distilled water) was used for the unwinding at 4 °C for 40 min. Electrophoresis was carried out at 4 °C for 30 min at 21 V and 300 mA. Neutralization of the chips was performed with 0.4 M Tris-HCl buffer at pH 7.5. SYBR gold nucleic acid gel staining (Invitrogen, cat. no. S11494) was used to stain the comets which were then fluorescently imaged at 4× magnification using an epifluorescence microscope (Nikon Eclipse 80i, Nikon Instruments, Melville, NY).