Pre-warmed PBS was slowly inflated into the lungs and withdrawn. The pooled sera were then centrifuged, and the supernatants were maintained at −70°C until use for ELISA. The cell pellets were resuspended and washed twice in PBS. The total cell numbers

were counted using a haemocytometer after RBC lysis using ACK lysis buffer (Invitrogen, Carlsbad, CA, USA). The BALF cell smears were prepared using a Cytospin apparatus (Hanil Science industrial Co., Gyeuangku, Inchun, Korea). The smears were then stained with Diff-Quik solution (Dade Diagnostics of Puerto Rico. Inc., Aguada, Puerto Rico) to determine the cell differentials, in accordance with RGFP966 chemical structure conventional Androgen Receptor antagonist morphological criteria. The results were calculated after three consecutive experiments. All animal studies were approved by the Animal Care and Use Committee of Pusan National University. After the mice were killed, the spleen, lung and lung draining lymph nodes were disrupted and treated with ACK hypotonic lysis solution (Sigma-Aldrich) for 2 min at room temperature for RBCs (red blood cells) lysis. After RBC lysis, the remaining cells were filtered with 100 μm mesh (Small Parts, Inc. Miramar, USA), and the cells were plated in 48 well plates as 5 × 106 cells/mL in RPMI 1640 with 10% foetal bovine serum and penicillin/streptomycin. For the CD3 stimulation

experiments, 0·5 μg/mL of CD3 antibody

(BD Pharmingen™, San Diego, CA, USA) was added to cell-plated wells. Plated cells were incubated for 72 h at 37°C in an atmosphere of 5% CO2. Following incubation, the culture media was harvested and stored at −20°C. The quantities of IL-4, IL-5, IL-13, IL-22, IL-17 and IL-17F in the BALF were determined using an enzyme Cobimetinib datasheet immunoassay, as previously described (22). Mouse lung epithelial cells (MLE12) were obtained from the American Type Culture Collection. Primary lung epithelial cells were isolated from C57BL/6 mice as described previously (22,24), after depletion with anti-CD32/CD16 and anti-CD45 antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were then treated with 0·01–1 μg/mL ES proteins. After 2 h of stimulation, the cells were collected and lysed, and the total RNA was extracted. Mouse embryonic fibroblast (MEF) cells were isolated from wild-type (WT) mouse, toll-interleukin 1 receptor (TIR) domain-containing adapter-inducing interferon-β (TRIF) knock out (KO), and Myeloid differentiation primary response gene (MyD) 88/TIR-domain-containing adaptor protein (TIRAP) KO C57BL/6 mouse foetuses 10 days after fertilization. Total RNA extracted using TRIzol reagent (Invitrogen Life Technologies, Milan, Italy) was used to generate cDNA using oligo-dT, random hexamers and SuperScript RT II (Invitrogen, Carlsbad, CA, USA).