Cells were cultured in DMEM (Sigma Aldrich, Taufkirchen, Germany) supplemented with 10% heat-inactivated endotoxin tested fetal calf serum (FCS, Cambrex, Verviers, Belgium), 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, and 2 mM L-alanyl-L-glutamine. Normal exocervical keratinocytes (NECK) were isolated from hysterectomy specimens by enzymatic digestion and cultured in KBM Gold medium (Lonza, Basel, Switzerland). Mycoplasma testing was performed monthly.
Immunohistochemical and Immunofluorescence analysis
Two-micrometer thick sections fixed on microslides were deparaffinized with xylene and hydrated using a diluted alcohol series. For immunhistochemistry sections were incubated with citrate buffer (10 mM, pH 7.0), for immunofluorescence in TE buffer (10mM Tris, 1mM EDTA pH9,0) and cooked for 10 min or 20 min, respectively for antigen retrieval and immersed in 3% H2O2 in TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.6) to block endogenous peroxidase activity. To reduce non-specific staining, each section was treated with 2.5% normal horse serum for 30min. Goat anti-CCL20 polyclonal antibody AF360 (1:100, R&D Systems, South Beloit, IL), mouse anti--smooth muscle actin (-SMA) monoclonal antibody 1A4 (1:500, Dako, Hamburg, Germany), rabbit anti IL-17 polyclonal antibody (1:1000, Abcam, Cambridge, UK), mouse anti-CD4 monoclonal antibody 4B12 (1:500, Leica Biosystems, Wetzlar, Germany) and mouse anti-CCR6 monoclonal antibody (1:1000, R&D Systems, South Beloit, IL) were used. For immunohistochemistry ImmPRESS Detection Kit (Vector Laboratories, Burlingame, USA) were used. For immunofluorescence Biotinylated Horse Anti-Mouse IgG with DyLight488 (Vector Laboratories) and TSA™ Kit #13, with HRP-Goat Anti-Rabbit IgG and Alexa Fluor® 546 Tyramide (Life Technology, Darmstadt, Germany) were used according to the manufactures instructions. Slides were evaluated with a DMI 6000B microscope (Leica, Wetzlar, Germany) and Microsoft Image Composite Editor program using standardized settings. To evaluate the number of infiltrating Th17 cells biopsies were co-stained for CD4 and IL-17 or CCR6 and IL-17. Five randomized pictures (200x) were taken per biopsy and the number of CD4/IL-17- or CCR6/IL-17-positive cells was counted.
Generation of conditioned media
For conditioned media cells were cultured at a density of 1 x 106/ml. After 24 h, fresh RPMI1640 medium (Sigma) plus supplements (10% heat-inactivated endotoxin-tested fetal calf serum (FCS; Cambrex, Verviers, Belgium), 100 U/ml penicillin, 100µg/ml streptomycin and 1mM sodium pyruvate) was added. Conditioned media were collected 24 h later.
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