Plaque reduction neutralization assay.

MDCK cells (MDCK.2 [ATCC CRL-2936]) were grown to confluence in 24-well plates. SP-D protein (4 μg/ml) was sequentially diluted 2-fold in d-PBS containing Ca²⁺ and Mg²⁺ (up to 0.25 μg/ml), and each dilution was mixed with an equal volume of virus corresponding to 20 to 30 plaques per well. Final SP-D concentrations ranged from 2 μg/ml to 0.125 μg/ml. Following preincubation at room temperature for 45 to 60 min, the mixed samples (100 μl total) were layered onto the MDCK cells. Each sample was tested in triplicate wells. A virus control without SP-D protein was included. After incubation at 37°C for 1 h, a plaque assay with staining after 48 h was performed.