Heat inactivated fetal bovine serum

MDCK cells (CCL-34) were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in minimum essential medium (Sigma-Aldrich Co. LLC) supplemented with 5% (v/v) heat-inactivated fetal bovine serum (Biosera, Kansas, MO, USA) and 50 μg/mL gentamicin sulfate solution (Fujifilm Wako Pure Chemical Corporation). Influenza A virus (IAV) strain A/Puerto Rico/8/1934 (H1N1; VR-1469) was obtained from the American Type Culture Collection. IAV was propagated at 37 °C using MDCK cells in serum-free medium (SFM; Thermo Fisher Scientific Japan K.K., Kanagawa, Japan) supplemented with acetylated trypsin (2 μg/mL) (Sigma-Aldrich Co. LLC) and gentamicin sulfate solution (50 μg/mL). The virus pellets were obtained according to some previous reports with minor modifications [11 (link),12 (link),13 (link)]. Briefly, the culture medium containing the propagated virus was centrifuged at 300× g for 5 min, the supernatant was then centrifuged at 13,000× g for 2 h, and the supernatant after centrifugation was discarded; the pellet was then re-suspended, and the solution was collected as a condensed viral solution.