Cell culture
Bovine mammary epithelial cells (BMECs; provided by Yangzhou University,
College of Animal Science and Technology Feed Engineering Technology
Research Center, China) were cultured in DMEM-F12 medium (ThermoFisher
Scientific, Waltham, MA, USA) containing 10% FBS (ThermoFisher
Scientific, Waltham, MA, USA), 1% penicillin and streptomycin (Beyotime
Biotechnology, Shanghai, China), 15 ng/mL EGF (PeproTech, Cranbury, NJ,
USA), 1% non-essential amino acids (ThermoFisher Scientific, Waltham,
MA, USA), and 1% insulin-transferrin-selenium (ThermoFisher Scientific,
Waltham, MA, USA) at 37℃ in an atmosphere of 5% CO2 and
95% air at 95% relative humidity. Briefly, BMECs (5 ×
105) were seeded onto six-well cell-culture plates in
the aforementioned medium for 48 h and were then cultured in DMEM-F12
medium containing 5% FBS, 1% penicillin and streptomycin, and 1% ITS
for 72 h to achieve a polarized and differentiated state. Before
treatments, the medium was changed into DMEM-F12 alone, and bothE. coli and LCZ were diluted by DMEM-F12. Cells were randomly
treated under one of four conditions as follows: (i) Control group (DMEM
alone); (ii) 1 × 103 CFU E. coli treatment for
3 h (E. coli group); (iii) 1 × 105 CFU LCZ
pretreatment for 5 h, followed by 1 × 103 CFU E.
coli treatment for 3 h (LCZ + E. coli group); and (iv) 1×
105 CFU LCZ treatment for 5 h (LCZ group). At 3 h
after pretreatment, the cells were washed three times with PBS
(ThermoFisher Scientific, Waltham, MA, USA) and exposed to E.
coli .