System microscope

For analysis of DNA damage after exposure to chemicals, alkaline comet assay was carried out, 21 with UV light exposure for 20 minutes as positive control. After comet assay, the gels were stained with DAPI (4′,6-diamidino-2-phenylindole) (100 μg/mL) and viewed using epifluorescent microscope (model BX51; Olympus System Microscope; Olympus, Southend-on-Sea, United Kingdom). Extent of DNA damage was determined by CometScore software (TriTek Corp, Sumerduck, Va), with analyses of densitometric and geometric parameters including percent tail DNA and olive tail moment (product of tail length and fraction of total DNA in tail).

Coverslips, 18 × 18 mm, were placed in a 3.5 cm diameter petri dish, seeded with 10 6 cells of each cell line culture and infected with the EAV Bucyrus strain at MOI 5. Infected cells were incubated as described above, then each coverslip was washed with phosphate-buffered saline (PBS) and stained with AO/EB (Sigma Aldrich, St. Louis, MO) (Kasibhatla et al. 2006) . Uninfected culture cell monolayers were used as negative controls and cultures where apoptosis was induced using 1 µg/ml staurosporine (Santa Cruz Biotechnology, Santa Cruz, CA) were used as positive controls. All tests were performed in duplicate.
A 1:10 dilution of a solution of 100 mg/ml AO and 100 mg/ml EB in PBS was added to each coverslip for 2 min, then the solution was removed and coverslips were mounted on a clean microscope slide and examined by 125 fluorescence microscopy (Model BHS; Olympus System Microscope, Bio Analítica, Buenos Aires, Argentina).