Atcc ccl 34

Influenza stock titers and viral load in ferret nasal lavage samples were determined by a standard focus forming assay on MDCK CCL-34 cells [67 (link), 68 (link)]. Briefly, serial dilutions of test sample were inoculated onto Mandin-Darby Canine Kidney (MDCK; ATCC^® CCL-34^™) cells and incubated in the presence of an Avicel (FMC BioPolymer) based overlay. Plates were then fixed and viral foci detected immunocytochemically via sequential incubation with anti-influenza A nucleoprotein clones A1 and A3, a HPR conjugated goat anti-mouse IgG (SeraCare), and TrueBlue^™ peroxidase substrate (SeraCare). Viral titers are expressed as focus forming units per mL (FFU/mL).

Madin-Darby canine kidney (MDCK) cells (ATCC CCL-34) and influenza A(H1N1) strain A/WS/33 (ATCC VR-825) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Complete growth medium for MDCK cells consisted of Eagle’s Minimum Essential Medium (EMEM) (ATCC) containing 10% fetal bovine serum (Hyclone Laboratories Inc, Logan, UT), 200 units/ml penicillin G, 200 μg/ml streptomycin (Invitrogen, Carlsbad, CA). MDCK cells were incubated at 35 °C in a humidified 5% CO₂ incubator until approximately 80% confluent. Propagation of influenza A(H1N1) [1.0×10⁷ TCID₅₀] and dilution in Viral Transport Media (VTM) consisting of Hank’s Balanced Salt Solution (1X HBSS; ThermoFisher Scientific) supplemented with 0.1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA), 100 units/ml penicillin G and 100 units/ml streptomycin (ThermoFisher Scientific), was performed as previously described (Blachere et al., 2011 (link)).

Professor Adolfo Garcia-Sastre (Icahn School of Medicine at Mount Sinai) provided Influenza A/Puerto Rico/8/34 (H1N1) virus. We maintained stock virus at −80 °C until grown for our enzyme-linked immunoassays (ELISA) and plaque assays. We purchased Madin-Darby canine kidney (MDCK) cells from ATTC (ATCC^® CCL-34^™). We stored MDCK cells at −80 °C until re-cultivated for the plaque assays. Immune Technology Corporation (AA 1-529) was the source for recombinant H1N1 hemagglutinin (His tag). We purchased all ELISA materials from BD Biosciences (San Jose, CA). We purchased Alhydrogel^® adjuvant 2% from INVIVOGEN (San Diego, CA). We suspended bLF in endotoxin- and preservative-free sterile saline (Vitality Medical, Cottonwood Heights, UT). The Food Science and Technology Institute, Morinaga Milk Industry Company, Ltd., Zama City, Japan provided a gift of 1 gm of purified, freeze-dried bovine lactoferrin powder. Biochemical reagents came from Scientific Fisher or Sigma-Aldrich.