Bronchial epithelial growth media

BEAS‐2B cells, which are virus‐transformed human bronchial epithelial cells, and primary normal human bronchial epithelial (NHBE) cells were respectively purchased from ATCC and Lonza and cultured according to the suppliers' recommendations. Briefly, BEAS‐2B cells (2.0 × 10⁵ cells/well) and NHBE (1.0 × 10⁴ cells/well) cells were cultured with serum‐free bronchial epithelial growth media (Lonza) to a confluence of 80%–100%, which usually took 3 days. Cells were afterwards stimulated with cytokines and/or TLR ligands for 24 h in a 24‐well culture plate unless otherwise specified. For inhibitory assays, BEAS‐2B cells were co‐incubated with inhibitors for 2 h before cytokine stimulation. To evaluate the activity of inhibitors, percent (%) inhibition at each concentration of the inhibitor was calculated using the following equation:
$\%\text{inhibition}=\left(1-A/B\right)\times 100,$ where A and B were culture‐media CCL5 concentrations of BEAS‐2B cells grown with poly(I:C) after pre‐incubation treatment with an inhibitor and a vehicle, respectively. We then performed a curve fitting analysis using the following equation:
$Y=\text{Bottom}+X\times \left(\text{Top}-\text{Bottom}\right)/\left(E{C}_{50}+X\right)$ and calculated the maximal inhibitory effect of inhibitors.

NHBE cells from two human donors (24-year-old and 69-year-old female Caucasians) were purchased from Lonza (Lonza Walkersville, Inc., Walkersville, MD, USA) and maintained in bronchial epithelial growth media (Lonza Walkersville, Inc., Walkersville, MD, USA). Cells at the third passage were grown into a confluent monolayer for subsequent infection experiments.

Primary small airway epithelial cells (SAECs) and normal human bronchial epithelial (NHBE) cells were cultured in bronchial epithelial growth media (Lonza Biosciences, Basel, Switzerland) and human MRC‐5 fibroblasts were grown in Eagle's medium (Sigma‐Aldrich, Gillingham, Dorset, UK), according to the suppliers' instructions. Epithelial cells were stimulated with a 1 in 5 dilution of CoMTb and MRC‐5 cells with a 1 in 50 dilution. Supernatants were harvested at 72 h for secretion analysis and mRNA extraction was performed at 24 h 14, 36.