Recombinant human il 8

Meningitic E. coli strain infection of primary hBMECs, as well as U251 cells and HUVECs, was performed in accordance with previously described methods61 62 (link). Briefly, E. coli overnight culture was resuspended and diluted in serum-free medium and added to the starved confluent primary hBMEC monolayer grown in 10-cm dishes at a multiplicity of infection of 10 (approximately 10⁸ colony-forming units per dish) to allow invasion at 37 °C for 3 h. For cytokine stimulation, recombinant human IL-8, MIP-2, GRO-α, IL-1β, IL-6 and TNF-α were purchased from Novoprotein Scientific (Shanghai, China) and used at a final concentration of 10 ng/mL to stimulate the primary hBMECs for 24 h. Finally, cells were washed three times with chilled PBS and subjected to RNA extraction by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), in accordance with the manufacturer’s instructions.

HUVEC cell mobility and migration was evaluated using a modified Boyden chamber (8-μm pore size; Merck Millipore). A total of 1 × 10⁵ HUVECs in 200 μl of serum-free medium were added to the top chamber, and 600 μl of serum-free medium, tumor culture medium supernatant or 50 ng/ml recombinant human IL-8 (Novoprotein) was added to the bottom chamber. Both chambers contained 0.5 μM SB225002 or 0.1% DMSO. After 24 h of migration, filters were rinsed with PBS, fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. The migrated cells on the underside of the whole transwell inserts were quantified by manual counting and photographed under a light microscope. The ability of HUVECs to form capillary-like endothelial tubes was determined in vitro. Briefly, a total of 1 × 10⁵ HUVECs were seeded on a Matrigel (Thermo Fisher Scientific) pre-coated 24-well plate and incubated in serum-free medium, tumor culture medium supernatant or 50 ng/ml recombinant human IL-8 with SB225002 (0.5 μM) or 0.1% DMSO for an additional 3-4 h. Photomicrographs from each well were captured and analyzed using ImageJ software, version 2.02 (National Institutes of Health, United States).