All ExPEC strains used in this study were isolated from extraintestinal tissues, among which PCN033 was a swine cerebrospinal fluid isolate [27 (link)]. E. coli strain RS218 (O18:K1:H7) was obtained also as a cerebrospinal fluid isolate from a case of neonate meningitis, which used as the positive control strain, and E. coli K12 strain HB101 was as the negative strain [13 (link), 55 (link)]. All E. coli strains were grown aerobically at 37°C in Luria–Bertani (LB) medium unless other specified.
The hBMEC cell line was kindly provided by Prof. Kwang Sik Kim in Johns Hopkins University School of Medicine [56 (link), 57 (link)], and was routinely cultured in RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum, 10% Nu-Serum, 2 mM L-glutamine, 1 mM Sodium pyruvate, nonessential amino acids, vitamins, and penicillin and streptomycin (100 U/mL) in 37°C incubator under 5% CO2 until monolayer confluence. In some experiments, confluent hBMEC was washed thrice with Hanks' Balanced Salt Solution (Corning Cellgro, Manassas, VA, USA) and starved in serum-free medium (1:1 mixture of Ham's F-12 and M-199) for 16–18 h before further treatment. As specified in some assays, cells were pretreated with various inhibitors prior to addition of the bacteria.
The hBMEC cell line was kindly provided by Prof. Kwang Sik Kim in Johns Hopkins University School of Medicine [56 (link), 57 (link)], and was routinely cultured in RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum, 10% Nu-Serum, 2 mM L-glutamine, 1 mM Sodium pyruvate, nonessential amino acids, vitamins, and penicillin and streptomycin (100 U/mL) in 37°C incubator under 5% CO2 until monolayer confluence. In some experiments, confluent hBMEC was washed thrice with Hanks' Balanced Salt Solution (Corning Cellgro, Manassas, VA, USA) and starved in serum-free medium (1:1 mixture of Ham's F-12 and M-199) for 16–18 h before further treatment. As specified in some assays, cells were pretreated with various inhibitors prior to addition of the bacteria.
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