Tnm fh

High Five cells (BTI-TN-5B1–4, Trichoplusia ni) were grown in serum-free Express Five media (Gibco) containing 10% L-Glutamine and 1% penicillin/streptomycin antibiotics mix. Sf9 cells (Spodoptera frugiperda) adapted from the cell line ATCC CRL-1711 were maintained in Trichoplusia ni medium—Fred Hink (TNM-FH, Gemini Bioproducts) supplemented with 1% penicillin/streptomycin antibiotics mix, 1% pluronic F-68 (Sigma-Aldrich), and 10% fetal bovine serum (FBS). In order to passage the baculoviruses, the media was switched to 3% TNM-FH (1% penicillin/streptomycin, 1% pluronic F-68, 3% FBS).

BTI-TN-5B1-4 (Trichoplusia ni, High Five) cells were passaged in Express Five medium (Gibco) supplemented with 1% l-glutamine (Gibco) and 1% penicillin/streptomycin antibiotic mix (100 U/ml of penicillin, 100 μg/mL streptomycin; Gibco). Sf9 (Spodoptera frugiperda) cells were passaged in Trichoplusia ni medium-Fred Hink (TNM-FH; Gemini Bioproducts) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% Pluronic-F68 (Sigma-Aldrich), and 1% penicillin/streptomycin antibiotic mix. To passage the baculoviruses in Sf9 cells, the medium was switched to TNM-FH medium containing 3% FBS, 1% Pluronic F-68, and 1% penicillin/streptomycin antibiotics. Madin-Darby canine kidney (MDCK) cells (ATCC CCL-34) were passaged in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% FBS, 1% penicillin/streptomycin antibiotic mix, and 1% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Gibco). The challenge virus, A/Singapore/GP1908/2015 (H1N1), was grown in 10-day-old embryonated chicken eggs (Charles River Laboratories), and titers were determined using a standard plaque assay (24 (link)). The virus is a reassortant virus containing the glycoproteins of A/Singapore/GP1908/15 (pH1N1, IVR-180) and the internal proteins of A/Texas/1/77 (H3N2). The virus was sourced from the National Institute for Biological Standards and Control.

Sf9 (Spodoptera frugiperda) insect cells were grown in Trichoplusia ni medium–Fred Hink (TNM-FH, Gemini Bioproducts, Sacramento, CA, USA) insect cell medium supplemented with 1% penicillin–streptomycin antibiotics mix (100 U/mL of penicillin, 100 µg/mL streptomycin, Gibco, Gaithersburg, MD, USA), 0.1% pluronic F-68 (Sigma-Aldrich, St. Louis, MO, USA), and 10% fetal bovine serum (FBS, Gibco). For passaging of the baculoviruses in Sf9 cells, TNM-FH insect medium (1% penicillin/streptomycin, 1% pluronic F-68, 3% FBS) was used. BTI-TN-5B1-4 (Trichoplusia ni, High Five) cells were passaged in serum-free Express Five insect cell medium (Gibco) containing 1% penicillin–streptomycin and 1% L-glutamine (Gibco). The challenge virus A/Singapore/GP1908/2015 (H1N1) was grown in 10-day-old embryonated chicken eggs (Charles River laboratories, Wilmington, MA, USA). Viral titers were determined by performing a standard plaque assay as previously described [31 (link)]. To purify the virus for the neuraminidase inhibition (NI) assay, A/Michigan/45/2015 was grown in 10-day-old eggs for 2 days at 37 °C, harvested, and purified using a 30% sucrose gradient in 1× NTE buffer (0.5 mM NaCl, 10 mM Tris-HCl pH 7.5, 5 mM ethylenediaminetetraacetic acid (EDTA)).