Cell Culture Protocols for Vero E6, Caco-2, IPEC-J2, and HEK 293T

Vero E6 cells (ATCC CRL-1586) were kindly provided by the Veterinary Medicine Research Center of the Da Bei Nong Group [82 (link)]. Caco-2 and IPEC-J2 (Guangzhou Jennio Biotech Co, Ltd., China). HEK 293T cells were purchased from ATCC (United States) [83 (link)]. Caco-2, and IPEC-J2, Vero and HEK 293T cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM from Life Technologies). All cells were supplemented with 10% fetal bovine serum (FBS, GIBCO), 16 mM HEPES (Life Technologies), and 100 μg/ml penicillin/streptomycin (Invitrogen) in a humidified atmosphere containing 5% CO₂ at 37°C. Cells were routinely seeded at a density of 2 × 10⁵/mL in 25 cm² plastic tissue culture flasks (Corning) and passaged every 3–4 days for a maximum of 30 passages and regularly tested for mycoplasma contamination.