Deae dextran

Postinfection IAV titers were determined by plaque assay on MDCK cells seeded in 6-well plates at 1 × 10⁶ cells per well. For drop infections, the viral supernatants were sampled from broken drops at 0 and 24 hpi. For bulk infections, viral supernatants were sampled from the tissue culture plate wells at 0 and 24 hpi. The supernatants were serially diluted in DMEM media with 1 mM HEPES (HyClone), 1× penicillin-streptomycin, 0.1% BSA, and 2 μg/mL of TPCK-trypsin. Overlay medium consisting of 2× MEM with 2× penicillin-streptomycin, 1 mM HEPES, 2 μg/mL of TPCK, and 3% carboxymethyl cellulose (MP Biomedical) with 0.2 mg/mL DEAE-dextran (MP Biomedical) was added, and well plates were incubated at 37°C for 4 days. Plaques were fixed with 10% buffered formalin (Fisher), washed with deionized (DI) water, and stained with 0.5% crystal violet (Thermo Fisher Scientific) for visualization.

Viral titers in the cell culture supernatants, tissue homogenates, and nasal washes were determined by plaque assay on MDCK cells. Tissues from infected mice were harvested at the indicated dpi and homogenized in 1mL of PBS/0.2% BSA (MP Biomedicals) and clarified of debris by centrifugation. Serially diluted supernatants were added to confluent monolayers of MDCK cells seeded in 12-well plates and overlayed with MEM media (1x MEM with 0.2% BSA, Avicel (FMC Biopolymers), 0.1% NaHCO3 (Sigma), 0.01% DEAE-dextran (MP Biomedicals)). After a 48h incubation at 37°C, the cells were fixed with 4% formaldehyde for 1h and plaques were visualized by staining with 0.1% crystal violet solution. Plaque assays for low pathogenic viruses were performed in the presence of 1μg/ml of TPCK-treated trypsin (Sigma).