Culturing MDCK, hCK, and Expi293F cells

Madin-Darby canine kidney (MDCK) cells (American Type Culture Collection; CCL-34) were maintained at 37 °C/5% CO₂ in minimum essential medium (MEM; Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and pen-strep mix (100 units/mL penicillin and 100 μg/mL streptomycin; Life Technologies). Humanized MDCK (hCK) cells [47 (link)] were maintained at 37 °C/5% CO₂ in Dulbecco’s Modified Eagle’s Medium-high glucose (DMEM; Sigma-Aldrich, St. Louis, MO, USA) containing 5% fetal bovine serum, pen-strep mix (100 units/mL penicillin and 100 μg/mL streptomycin), 5 µg/mL blasticidin (InvivoGen, San Diego, CA, USA), and 2 µg/mL puromycin dihydrochloride (Life Technologies). Expi293F cells (Thermo Fisher Scientific) were maintained at 37 °C/8% CO₂ in Erlenmeyer cell culture flasks (Corning, Tewksbury, MA, USA) containing Expi293 Expression Medium (Thermo Fisher Scientific). A/Narita/1/2009 (H1N1)pdm09 virus [19 (link)] and A/Narita/1/2009 (H1N1)pdm09-derived F11 escape mutants (C1 and G6 [18 (link)]) were propagated in MDCK cells.