Trypsin tolylsulfonyl phenylalanyl chloromethyl ketone

Madin–Darby canine kidney (MDCK) cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (HyClone Laboratories, Logan, UT, USA) containing 10% fetal bovine serum (HyClone Laboratories), 100 U/mL penicillin, and 100 μg/mL streptomycin. Right lung homogenates from individual mice were prepared and diluted 10-fold in DMEM and added to a monolayer of MDCK cells in semisolid agar containing 0.5 μg/mL trypsin-tolylsulfonyl phenylalanyl chloromethyl ketone (TPCK) (Sigma, Beijing, China). The cultures were incubated at 37°C and 5% CO₂ for 60–72 h, fixed, and stained with 1% crystal violet. Plaque-forming units (PFUs) were then counted.

The human embryonic kidney cell line HEK293 was kindly donated by Prof. Dr. Duncan R. Smith of the Institute of Molecular Biosciences, Mahidol University. The HEK293, HEK293T, and A549 cells (CRL-3216, CCL-185; ATCC, Manassas, VA, USA) were cultivated in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Invitrogen) at 37 °Celsius ( °C) in a humidified incubator with 5% carbon dioxide (CO₂). The Madin-Darby Canine Kidney (MDCK; CCL-34, ATCC) cell line was cultivated in Minimum Essential Medium (MEM; Gibco, Invitrogen) supplemented with 10% heat inactivated FBS at 37 °C in a humidified incubator with 5% CO₂.
The IAVs A/Nonthaburi/102/2009(H1N1) were propagated in MDCK cells with MEM in the presence of tolylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-trypsin (Sigma-Aldrich Corporation, St. Louis, MO, USA) at a concentration of 1 µg ml⁻¹ (MEM+TPCK-trypsin). The supernatants containing virus were harvested by centrifugation to remove cell debris and stored at −80 °C until use. The viral titer in MDCK cells was determined by plaque assay.