Comet assay kit for single cell gel electrophoresis

The sources of the chemopreventive agents used in this study are listed in Table 1. Prior to conducting the work, all agents were assessed for purity using LC-UV-MS with a Shimadzu (Kyoto, Japan) IT-ToF high resolution hybrid mass spectrometer equipped with reversed phase HPLC, electrospray and an in-line UV absorbance array detector (Table 1). HPLC-grade methanol and acetonitrile were purchased from Sigma-Aldrich (St. Louis, MO) or Thermo-Fisher (Waltham, MA). Distilled water, prepared by a Milli-Q water purification system from Millipore (Milsheim, France) was used throughout the study. Endonuclease III from E.coli, recombinant, Histopaque-1077, Hank’s balanced salt solution (HBSS), dimethylsulfoxide (DMSO), phosphate buffered saline (PBS), and hydrogen peroxide (H₂O₂) were purchased from Sigma-Aldrich. Trizol reagent was purchased from Invitrogen (Life Technologies, Foster City, CA). SYBR Gold nucleic acid stain was from Life Technologies (Carlsbad, CA), Trypan blue solution, 0.4% was purchased from Gibco (Grand Island, New York). Comet assay kit for single cell gel electrophoresis was purchased from Trevigen (Gaithersburg, MD). RT² Profiler custom made PCR Array: CAPF12480 related to dog oxidative stress was purchased from QIAGEN-Frederick (SABiosciences, Valencia, CA) (Table 5). All other chemicals were of analytical grade.

Cisplatin genotoxicity in treated and untreated HL-60 cells was analyzed by alkaline single cell gel electrophoresis (Comet) assay as described earlier [23 (link)] with few modifications using Comet assay kit for single cell gel electrophoresis from Trevigen (Gaithersburg, MD, USA). All the precautions were taken to avoid the UV light effect on DNA. Low melting agarose was melted in boiling water and cooled down to 37°C. For each treatment, 75 µL of the agarose and cells mixtucre (ratio of 1:10) was placed on comet slides and solidified at 4°C. Cells were lysed with lysis solution for 30 min at 4°C, followed by denaturing the DNA with alkaline solution for 40 min at 37°C. The prepared slides were subjected to electrophoresis (1 volt/cm) for 10 min in TBE (Tris borate EDTA) buffer. After the electrophoresis, cells were fixed with 70% ethanol followed by staining with SYBR green. Epifluorescent microscope (Olympus BX51 TRF, USA) was used to observe the comet slides. The data was evaluated using the DNA damage analysis software (Loats Associates Inc., USA).