Dmem f12 culture medium

Primary enterocytes were prepared using a modified method, as described elsewhere [41 (link), 42 (link)]. Small intestines were opened longitudinally and cut into small pieces (~ 1 mm × 1 mm). After being washed, the tissues were soaked on ice for 90 min in Ca²⁺ and Mg²⁺-free Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, Inc.) containing 3 mM EDTA and 1 mM DTT. The tissues were shaken vigorously to detach crypts, which were pelleted by centrifugation at 150 × g for 3 min at 4 °C. The pellets were washed twice with ice-cold Ca²⁺ and Mg²⁺-free HBSS and resuspended in DMEM/F12 culture medium (BioWhittaker, Walkersville, MD), which was supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 × insulin-transferrin-selenium (Invitrogen), 20 mM HEPES (pH 7.4), 0.25 μg/ml amphotericin B, 1 μg/ml fibronectin, and 1 μg/ml hydrocortisone. Twenty-four-well plates were coated with collagen I and poly-L-lysine 1 h before the cells were seeded. The plates were seeded with approximately 200 to 300 organoids/cm² and incubated at 37 °C under a 5% CO₂ atmosphere. Three days after being seeded, the cells were infected with JEV at different MOI (0.01, 0.1, and 1.0).