N tosyl l phenylalanine chloromethyl ketone trypsin

Influenza A virus (H1N1 strain PR8), which was a kind gift from Dr. Genhong Cheng (University of California, Los Angeles), was propagated in the allantoic cavities of 11‐day‐old specific pathogen‐free embryonated chicken eggs at 35°C. Freshly collected allantoic fluids were clarified by low‐speed centrifugation at 72 h postinoculation and then stored in small aliquots at −80°C. The virus titers were determined using a plaque‐forming assay in monolayers of Madin‐Darby canine kidney (MDCK) cells.
For the TCID₅₀ assay, the IAV virus sample from infected HT‐29 cells was diluted in DMEM containing 1 μg/ml N‐tosyl‐L‐phenylalanine chloromethyl ketone‐trypsin (Sigma), 0.3% bovine albumin (Sigma), 1% penicillin/streptomycin, and 25 mM HEPES buffer (Gibco) across a 96‐well tissue culture plate with MDCK cells. After 48–72 h, TCID₅₀ titer per 100 μl was determined using the Reed‐Muench method (Lei et al, 2021 (link)).

VLPs harboring BlaM1 fusion proteins were produced essentially as described before (Tscherne et al., 2010 (link)). Briefly, HEK 293T cells seeded onto poly-L-lysine–coated (Sigma-Aldrich) 6-well plates were transfected in Opti-MEM (Gibco) with 2.5 µg BlaM1, 500 ng pCAGGS-WSN-HA, 1.125 µg pCAGGS-WSN-NA, 250 ng pCAGGS-WSN-M2, and 300 ng pCAGGS-IFITM3, pCAGGS-IFITM1, or pCAGGS per well using ViaFect (Promega) as the transfection reagent (2.5 µl ViaFect/µg DNA). Medium was exchanged 8 h after transfection. VLPs were harvested 72 h after transfection and treated with 6 µg/ml N-tosyl-L-phenylalanine chloromethyl ketone trypsin (Sigma-Aldrich) for efficient HA cleavage. For infection of MDCKII, MDCK control, or MDCK-IFITM3 cells, IAV-VLP input was normalized by Western blotting for BlaM1 using the mouse monoclonal anti-IAV M1 (HB-64; American Type Culture Collection) antibody. At 4 h after infection, cells were harvested by trypsinization and incubated with the fluorogenic substrate CCF2-AM (Thermo Fisher Scientific). Cells were analyzed on a FACSVerse System (BD Biosciences) and dead cells were excluded by a live/dead staining (LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit; Thermo Fisher Scientific).