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After treatment with erythrocyte-lysis-buffer (incl. NH4Cl, KHCO3 and EDTA) for 60 s, cells were washed twice in X-vivo medium and then cryoconserved following a standardized freezing procedure using cryomedium containing RPMI, FCS (20%) and DMSO (10%), respectively (Sigma-Aldrich, Munich, Germany). The other part of the collected tissue was sent to the Department of Pathology, University of Ulm, Germany, for hematoxylin�Ceosin stain. IL-5, IFN-��, TGF-��1, and TGF-��2 levels in nasal lavages were measured with the Quantikine? Kit (R&D-Systems, Wiesbaden, Germany) complying with the manufacturer��s recommendations. The detection limit for IL-5 was 3.0?pg/ml, 8.0?pg/ml for IFN-��, 4.61?pg/ml for TGF-��1, and 7.0?pg/ml for TGF-��2. Spiking controls performed for IL-5 and IFN-�� in advance by our group ALK (4) obtained loss of recovery selleck compound for TGF-��1 was 2.4-fold and for TGF-��2 1.4-fold. ECP and total IgE levels were measured using an automated fluorescence-enzymeimmunoassay (UniCAP100 Diagnostic System, Phadia, Freiburg, Germany) according to the manufacturers�� recommendations. The limit of detection was 0.7?��g/l for ECP and 2 kU/l for total IgE. Dilution was 5-fold for ECP. Total IgE was measured undiluted. Hematoxylin-eosine stained tissue slices were analysed in high-power fields (HPF) at 40?��? objective magnification (10?��? ocular). Eosinophils were identified and counted in 10 HPF per slice. For subsequent analysis, mean cell number per specimen was determined. PBMC as well as single cell suspensions from tissue samples were thawed and incubated with FcR blocking reagent (Miltenyi Pazopanib Biotech, Bergisch Gladbach, Germany). Dendritic cells were differentiated into myeloid (mDC) and plasmacytoid (pDC) subtypes (Fig.?1) with the MACS? dendritic cell enumeration kit (Miltenyi Biotech, Bergisch Gladbach, Germany). For each specimen, two samples with at least 1?��?106 PBMC (blood) or 1?��?106 separated cells (tissue) were needed. MDC and pDC were detected in one sample by staining with Anti-BDCA-1-PE and Anti-BDCA-2-FITC, respectively. The other sample was stained with FITC- and PE-conjugated isotype control monoclonal antibodies. Both samples were simultaneously stained with CD19-PE-Cy5 and CD14-PE-Cy5 for exclusion of B cells and monocytes. All monoclonal antibodies were supplied as premixed cocktails (Anti-BDCA and Control Cocktail). Simultaneously to antibody staining, samples were stained with a fluorescent cell-impermeant dye (dead cell discrimination).