Cell Culture
BEAS-2B cells and primary normal human bronchial epithelial cells (NHBE
cells) were purchased from ATCC (Manassas, VA, United States) and Lonza
(Tokyo, Japan), respectively, and were cultured according to
manufacturer recommendations. BEAS-2B cells are virus-transformed human
bronchial epithelial cells. Briefly, BEAS-2B cells (2.0 ×
105 cells/well) and NHBE (1.0 × 104cells/well) cells were cultured with serum-free Bronchial Epithelial
Growth Media (BEGMTM, Lonza) to a confluence of
80-100%, which usually took 3 days. Cells were afterwards stimulated
with cytokines and/or TLR ligands for 24 hours in a 24-well culture
plate unless otherwise specified. For inhibitory assays, BEAS-2B cells
were co-incubated with inhibitors for 2 hours prior to cytokine
stimulation. To evaluate the activity of inhibitors, percent (%)
inhibition at each concentration of the inhibitor was calculated using
the following equation:
% inhibition = (1-A/B) x 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 = Bottom + X * (Top - Bottom) / (EC50 + X)
and calculated the maximal inhibitory effect of inhibitors.