Tpck treated trypsin

All influenza viruses were propagated in MDCK cells and specific pathogen-free (SPF) embryonated chicken eggs. Monolayers of MDCK cells were washed with phosphate-buffered saline (PBS) and incubated with the respective virus at a multiplicity of infection (MOI) of 0.001 at 37°C. After 1 hour, the inoculum was aspirated, cells were washed twice and incubated at 37°C with DMEM without serum supplemented with tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (1 μg/ml; Worthington Biomedical Corporation, Lakewood, NJ). 48 hr postinfection (p.i.) virus was recovered from supernatants. For SPF eggs, 0.2 ml stock influenza virus at 1x10³ TCID₅₀ was injected into 11-day-old SPF fertile chicken eggs. The eggs were incubated in a stationary incubator at 35°C. After 72 hr incubation, eggs were cooled at -20°C for 30 min, then clear allantoic fluid was collected. For viral titration, plaque assays were performed as described [60 (link)]. Briefly, 1.2 × 10⁶ MDCK cells/ml were plated in 6-well plates. MDCK cells were washed twice with DMEM without serum, then serial dilutions of virus were adsorbed onto cells for 1 hr. Cells were then covered with DMEM 2×Avicel RC591 NF mix (FMC Biopolymer, Philadelphia, PA) supplemented with TPCK-treated trypsin (1 μg/ml). Crystal violet staining was performed 72 hr p.i., and visible plaques were counted.