Influenza Virus Plaque Assay Optimization

One hour after infecting the cell monolayers with 30–50 plaque forming units of the virus in 1 ml of maintenance medium without trypsin, we removed the virus inoculum, covered the cells with 3 ml of the different overlay media and incubated cultures at 35°C in 5% CO₂ atmosphere. In the case of MC and Avicel overlays, care was taken not to disturb the plates during the incubation period in order to avoid formation of non-even plaques. After three days of incubation, we removed the overlays and fixed the cells. Agar overlay was removed using metal spatula; MC, Avicel, and liquid overlays were removed by suction. The cells were fixed with 4% paraformaldehyde solution in MEM for 30 min at 4°C and washed with PBS. All subsequent treatments of the cells were performed at room temperature. We permeabilized the cells and simultaneously blocked residual aldehyde groups by incubating the cells for 10–20 min with 1 ml/well of solution containing 0.5 % Triton-X-100 and 20 mM glycine in PBS. We immuno-stained virus-infected cells by incubating for 1 hr with monoclonal antibodies specific for the influenza A virus nucleoprotein (kindly provided by Dr. Alexander Klimov at Centers for Disease Control, USA) followed by 1 hr incubation with peroxidase-labeled anti-mouse antibodies (DAKO, Denmark) and 30 min incubation with precipitate-forming peroxidase substrates. Solution of 10% normal horse serum and 0.05% Tween-80 in PBS was used for the preparation of working dilutions of immuno-reagents. We washed the cells after the primary and secondary antibodies by incubating them three times for 3–5 min with 0.05% Tween-80 in PBS. As peroxidase substrates, we employed either ready to use True Blue™ (KPL) or solution of aminoethylcarbazole (AEC, Sigma) (0.4 mg/ml) prepared in 0.05 M sodium acetate buffer, pH 5.5 and containing 0.03% H₂O₂. Stained plates were washed with tap water to stop the reaction and dried. In the case of True Blue staining, which is relatively unstable in water solutions, plates were dried inverted in order to minimize bleaching. Stained plates were scanned on a flat bed scanner and the data were acquired by Adobe Photoshop 7.0 software.
As an alternative to immuno-staining, in some experiments we revealed plaques as areas of destroyed cells. To this end, after removing the overlays, we stained the cells with 1% crystal violet solution in 20% methanol in water.