PHCEC and PHKF cells at 90% confluence were treated with TLR1-9 ligands (Invivogen) (San Diego, CA, USA) for 16 h with cell culture medium only as negative control. The various TLR ligands and their concentrations used were: TLR1/2 (Pam3CSK 4 1µg/ml), TLR2 (HKLM 108 cells/ml), TLR3 ((Poly(I∶C) 10 µg/ml), TLR4 (LPS 1µg/ml), TLR5 (Flagellin 1 µg/ml), TLR6 (FSL1 1 µg/ml), TLR7 (Imiquimod 1 µg/ml), TLR8 (ssRNA40 1µg/ml) and TLR9 (ODN2006 5 µM).
Cell supernatants were collected and multiplex bead assay analyzer (Luminex 100; Luminex Corporation, Austin, TX) was performed with a human cytokine 30-Plex kit (Invitrogen, Camarillo, CA) according to manufacturer's protocol and as previously described [26] (link). The panel included EGF, Eotaxin, FGF-basic, G-CSF, GM-CSF, HGF, IFN-α, IFN-γ, IL-1RA, IL-1β, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40/p70, IL-13, IL-15, IL-17, CCL2, CXCL9, CCL3, CCL4, CCL5, TNF-α, and VEGF.
Cell supernatants were collected and multiplex bead assay analyzer (Luminex 100; Luminex Corporation, Austin, TX) was performed with a human cytokine 30-Plex kit (Invitrogen, Camarillo, CA) according to manufacturer's protocol and as previously described [26] (link). The panel included EGF, Eotaxin, FGF-basic, G-CSF, GM-CSF, HGF, IFN-α, IFN-γ, IL-1RA, IL-1β, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40/p70, IL-13, IL-15, IL-17, CCL2, CXCL9, CCL3, CCL4, CCL5, TNF-α, and VEGF.