Modulation of nasopharyngeal innate defenses by viral coinfection predisposes individuals to experimental pneumococcal carriage

Glennie, S; Gritzfeld, J F; Pennington, S H; Garner-Jones, M; Coombes, N; Hopkins, M J; Vadesilho, C F; Miyaji, E N; Wang, D; Wright, A D; Collins, A M; Gordon, S B; Ferreira, D M

Pharyngeal epithelial cells and inflammation of epithelium. Detroit 562 pharyngeal epithelial cells (ATCC-CCL-138) were grown and maintained at 90-100% confluence in T75 flasks (BD) in Eagle's minimal essential media (EMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (Invitrogen, Paisley, UK), 2 mM L-glutamine (Sigma-Aldrich), 40 U ml À 1 penicillin, 40 mg ml À 1 streptomycin, and 80 mg ml À 1 neomycin (Sigma-Aldrich) (complete media). Before adherence experiments, cells were released using EDTA/trypsin (Invitrogen), washed with media, and a suspension of cells were seeded at 1 Â 10 5 per ml in complete media in a 24-well plate (1 ml per well). Cells were ready for use in adherence experiments 3 or 4 days after seeding (90% confluence). For inflamed epithelium, cells were stimulated with interleukin-1b, tumor necrosis factor, and interferon-g (50 ng ml À 1 each) in 500 ml of complete EMEM for 16-18 h at 37 1C 5% CO 2 . Cells were then washed 3 times with warmed PBS and 500 ml of EMEM without antibiotics was added to each well before addition of pneumococci.