2.20. Poly (ADP-Ribose) Polymerase-1 (PARP-1) Cleavage Assay

PARP-1 cleavage was assessed using rabbit-cleaved PARP-1 antibody (Santa Cruz Biotechnology Inc., SC-194C1439, Paso Robles, CA, USA). Cells infected with rotavirus Wt1-5 (MOI 0.1 to 6) in RPMI culture medium without FBS were harvested at 24 h.p.i., fixed in cold absolute ethanol for 30 min, collected by centrifugation at 400× g for 5 min and washed twice with PBS-T containing 1% BSA. Cells were plated onto coverslips and incubated with cleaved PARP-1 antibody (1 mL/mL) in blocking buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 0.3% [v/v] Tween 20 and 5% skimmed milk) for 1 h at RT. After washing twice with PBS-T, the cells were incubated with secondary donkey anti-rabbit FITC-conjugated antibody (0.88 mg/mL, Santa Cruz SC-2024, Paso Robles, CA, USA) in PBS containing 1% BSA for 40 min at 37 °C in the dark, and then, washed twice with PBS before treatment with rabbit hyperimmune serum against rotavirus structural proteins. Cells were incubated for 1 h at 37 °C and washed twice with PBS before the addition of secondary goat anti-rabbit Alexa Fluor 568-conjugated antibody (0.88 mg/mL, Santa Cruz Biotechnology Inc., SC-2780, Paso Robles, CA, USA) in PBS containing 1% BSA and incubation for 40 min at 37 °C in the dark. DAPI (4′,6-diamidino-2-phenylindole) (0.5 µM) was added followed by incubation for 30 min at 37 °C in a humidified chamber protected from the light. Fixed and permeabilized cells were treated with recombinant DNase I, grade I (3 U/mL) in reaction buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂ and 1% BSA) for 10 min at RT to induce hydrolysis of phosphodiester linkages to generate positive control cells. Cells treated with H₂O₂ (1 mM) and non-infected cells were also used as a control. Positive cells for PARP-1 cleavage and for structural rotavirus antigens were recorded and expressed as a percentage of the total cell population observed. Representative photographs were taken using a Nikon C1 confocal laser scanning microscope. The images were obtained with the Nikon NIS-Elements Advanced Research software and analyzed with the ImageJ 1.44p Java 1.6.0_20 (32-bit) software (Nikon, Tokyo, Japan).