Influenza Virus Plaque Assay in MDCK Cells.

MDCK cells (ATCC) were grown to 70 to 80% confluency in DMEM + 0.1mM nonessential amino acids + 1mM sodium pyruvate + 50 U/mL penicillin and 50 µg/mL streptomycin + 20 µg/mL gentamycin + 10% fcs in 6-well tissue culture plates in 37 °C + 5% CO₂. The medium was removed and the cells rinsed 2× with 0.3% BSA in DMEM, then 100 µL 10-fold serial dilutions (0.3% BSA in DMEM as diluent) of virus samples were adsorbed for 1 h at 37 °C + 5% CO₂ (300 µL diluent was added to each well during the adsorption to prevent drying of cell sheet). The inoculum was removed, the cells rinsed once with PBS, and then overlaid with 2 mL L-15 medium + 1 µg/mL TPCK-treated trypsin + 0.5% agarose. The plates were incubated for 48 h then stained with 0.3% crystal violet + 5% isopropanol + 5% ethanol in H₂O for 20 min following removal of the overlay and 30-min fixation in 90% ethanol. The cells were rinsed with H₂O and the plaques enumerated.