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The degree of airway inflammatory cell infiltration was scored in a double-blind fashion by two independent investigators. Lung lesions were scored semiquantitatively as described by other researchers [13]. The severity of inflammation was evaluated by assigning a value of 0 point for normal; 1 point for few cells; 2 points for a ring of inflammatory cells 1 cell layer deep; 3 points for a ring of inflammatory cells 2 to 4 cells deep; 4 points for a ring of inflammatory cells of >4 cells deep. Bronchoalveolar lavage fluid (BALF) was obtained by instilling and collecting two aliquots of 1?ml each of PBS through an adapter cannula inserted through rings of the exposed trachea of euthanized mice 24?h after final challenge with OVA. BALF was pooled to obtain one sample for each mouse. Erythrocytes were lysed, and the remaining [http://www.selleckchem.com/products/3-deazaneplanocin-a-dznep.html 3-deazaneplanocin A ic50] cells were cytocentrifuged 2500?rpm for 5?min. Total cell numbersin the BALF were determined using a standard hemocytometer. Differential cell counts were performed based on standard morphological and staining characteristics of at least 250 cells per sample. Supernatant was stored at ?80?��C. All slides were characterized [https://en.wikipedia.org/wiki/Thiram thiram] by a single blinded examiner to eliminate bias. Cytokine concentrations in BALF were measured with commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions. ELISA kits used for the measurement of IFN-��, IL-5, and IL-10 were purchased from Sizhengbai (Beijing, China), ELISA kits for detection of IL-4 and TGF-�� was purchased from Xinbosheng (Beijing, China), and the IL-17A and IL-13 detection ELISA kits were purchased from Bender. The mediastinal lymph nodes (MLN) were removed and forced through a 70?��m cell filter (BD, Bedford, MA, USA) to obtain single cell suspensions. Single cell suspensions in MLN were stained for surface-associated CD4(anti-CD4-FITC, BD Pharmingen, USA), CD3(anti-CD3-CyTM7, BD Pharmingen, USA), CD25(anti-CD25-PE, e Bioscience, USA), then fixed, permeabilized and stained for intracellular IFN-��(anti-IFN-��-PerCP-CyTM5.5,-BD [http://www.selleckchem.com/products/erastin.html Erastin datasheet] Pharmingen, USA), IL-17A (anti-IL-17A-PE, BD Pharmingen, USA), IL-4(anti-IL-4-APC, BD Pharmingen, USA) and Foxp3 (anti-Foxp3-PE-Cy5, e Bioscience, USA) and analyzed by flow cytometry (FACS Canto, BD Biosciences, USA). Results were analyzed using GraphPad Prism (version 5.0; GraphPad, La Jolla, CA) and expressed as mean?��?s.e.m. Results were interpreted using either one-way analysis of variance and Tukey's post hoc test, or two-way analysis of variance and Bonferroni's post hoc test. Differences were considered statistically significant when P?

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Brakearies94 : Page créée avec « The degree of airway inflammatory cell infiltration was scored in a double-blind fashion by two independent investigators. Lung lesions were scored semiquantitatively as d... »