M. hyopneumoniae ATCC 25095 was purchased from the American Type Culture Collection (ATCC) and cultivated in a mycoplasma medium (Basal Media, Shanghai, China) at 37°C. To estimate the numbers of CFU in the cultures, serial dilutions were plated on a modified pleuropneumonia‐like organism (PPLO) medium containing 1.5% agarose (V2111; Promega) and incubated at 37°C. CFU was counted 7–10 days later using a microscope (Vogl et al., 2008 (link)). M. hyopneumoniae was pelleted by centrifugation at 10,000g for 10 min and resuspended at 1.0 × 106 CFU/ml in phosphate buffered saline (PBS).
PTECs were prepared from the tracheas of two 5‐week‐old specific‐pathogen‐free (SPF) piglets using previously described protocols (Wang et al., 2018 (link)). Human embryonic kidney cells (HEK293T) were purchased from ATCC. PTECs and HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat‐inactivated fetal bovine serum (FBS), 1% antibiotics (100 μg/ml streptomycin and 100 U/ml penicillin; Gibco), and 10 mM HEPES (Invitrogen). The cells were incubated at 37°C in 5% CO2.
