Adhesion assay
The BMECs were washed with PBS three times, and one milliliter
triton-X-100 (0.5% V/V, Solarbio Life Science, Beijing, China) was
added in each well after incubation with LCZ or E. coli . The
cells were scraped off, and the lysate was transferred to a 1.5-mL
centrifuge tube after 10 min of stabilization at room temperature.E. coli and LCZ were plated on TSA and MRS agar, respectively,
after serial dilution and were quantified by determination of CFU.
Measurement of the
trans-epithelial
electric resistance
To measure the trans-epithelial electric resistance (TEER) of monolayer
BMECs, BMECs were seeded to confluence on transwell inserts (membrane
area 0.33 cm2, pore size 0.4 μm, Corning, NY,
USA)
and a polarized and differentiated state was achieved within 5 days
after seeding. TEER was measured immediately after different treatments
with a Millicell Electrical Resistance System-2 (Millipore, MA, USA).
Measurements of TEER were made three times per well and expressed as
Ω∙cm2 after subtracting the filter resistance
value.
The results of TEER were expressed as change of TEER (TEER of monolayer
cells after treatments - TEER of monolayer cells before treatments).