Influenza MDCK plaque assay

MDCK cells (ATTC, Manassas, VA) were seeded on six-well tissue culture plates at 4 × 10⁵ cells/well in 2 ml of MEM with Earle’s salts + 0.1 mM non-essential amino acids + 1 mM pyruvate + 50 U/ml penicillin + 50 µg/ml + streptomycin + 20 µg/ml gentamicin + 10% fetal calf serum and incubated at 37 °C, 95% relative humidity, and 5% CO₂. When cells reached 80–90% confluency (≈3 days) the cells were rinsed 2× with 1 ml 0.3% BSA in DMEM and 0.3 ml was added to prevent drying of the cells. Influenza samples were prepared, as described above, 10-fold serially diluted in 0.3% BSA in DMEM and kept on ice. Diluted samples were added to appropriate wells, 100 µl/well, and adsorbed for 1 h at 37 °C, 95% relative humidity, and 5% CO₂ while gently rocking. The samples were aspirated and the cells rinsed 1× with PBS + 50 U/ml penicillin + 50 µg/ml + streptomycin. 2× L-15 (Sigma Aldrich) with 25 mM HEPES + 0.15% NaHCO₃ + 100 U/ml penicillin + 100 µg/ml + streptomycin + 0.5 µg/ml gentamicin + 2 µg/ml TPCK-trypsin (Sigma Aldrich) was combined with an equal volume of liquefied 1.0% agarose in water then 2 ml/well was dispensed into the culture wells. After incubating the plates for 2 days at 37 °C, 95% relative humidity, and 5% CO₂ the L-15/agar was removed and the cells fixed with 2 ml/well 90% ethanol for 30 min on an orbital shaker at room temperature. The ethanol was replaced with 2 ml/well 0.3% crystal violet (Sigma Aldrich) in 5% isopropanol + 5% ethanol in water and incubated for 20 min at room temperature on an orbital shaker. Finally, the crystal violet was removed, the cells rinsed with 2 ml/well of water, and the cultures allowed to air dry prior to counting the virus plaques.