Quantification of Influenza A Virus Titer

Purified influenza A/PR/8/34 (PR/8) virus was obtained from Charles River, diluted 1:10 in sterile PBS, aliquoted and stored at -80°C. PR/8-OVA [22 (link)] was provided by Adolfo García-Sastre (Icahn School of Medicine at Mount Sinai, New York). Viral titers were determined by plaque forming assay using MDCK cells [57 (link)]. One day before the assay, 1x10⁶ cells/well were seeded in 6-well plates and grown overnight in Dulbecco’s Modified Eagle Medium (DMEM; Sigma), supplemented with 10% fetal calf serum (FCS; Sigma), 100 U/ml penicillin and 100 μg/ml streptomycin, to obtain monolayers with >90% confluency. Cells were then washed with PBS and serial 1:10 dilutions of virus in DMEM (without FCS and antibiotics) were added to the cells for 1 hour at 37°C (each dilution in duplicates). Inoculates were aspirated and overlay medium, consisting of DMEM with antibiotics supplemented with 0.005% DEAE-Dextran (Sigma), 0.2% endotoxin-free bovine serum albumin (Fisher Scientific), 0.2% TPCK-treated trypsin (Sigma) and 1% agarose (Biozym), was added. 3 days later, plaques were visualized by staining with 0.03% neutral red (Sigma) for 3 hours and quantified.