At the end of the incubation time, the reaction was stopped by the addition of PBS supplemented with 5% FCS. Subsequently,

the fragments were incubated with DNase I (50 U/ml) (Invitrogen) for 40 min at 37°. Finally, the cell suspensions were collected through a gauze mesh and washed with cold PBS. DCs were labelled with carboxyfluorescein succinimidyl www.selleckchem.com/products/Dasatinib.html ester (CFSE; 5 μm) for 40 min at 37°. Cells were extensively washed and re-suspended in PBS. DCs (1 × 106) were injected i.t. into BALB/c mice. Six hours later, lung tissues were collected and processed as described above. The presence of CFSE-labelled DCs in the lung suspensions was analysed by flow cytometry. A week after the treatment of allergic mice with PBS, DCs or DCHISs, lungs were washed via a tracheal tube with PBS. Cells were washed and leucocyte counts were determined by optical microscopy. Cytospin slides were stained with toluidine to determine the percentages of eosinophils. Cell 3-deazaneplanocin A datasheet staining was performed using the following monoclonal

antibodies (mAbs): anti-CD11c, anti-CD8α, anti-CD4, anti-CD8, anti-CD11b and anti-GR1 [conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE) or peridinin chrorophyl protein complex] (BD Pharmingen, San Diego, CA). The data were collected using a FACSCalibur (Bs.As., Argentina) flow cytometer and analysed using the CellQuest program (BD Biosciences; Bs.As., Argentina). Serum samples were obtained from mice at the end of experiments by cardiac puncture. OVA-specific IgE antibodies were detected using plates coated overnight with 1 μg/ml OVA in sodium carbonate buffer (pH 9·5; Sigma-Aldrich). Plates were treated with Tween 0·5% in PBS (TPBS) supplemented with 1% bovine serum albumin (BSA) for 2 hr at room temperature. Serial dilutions of sera were added and, after 2 hr, the plates were washed three times with TPBS and an appropriate dilution of biotinylated

detection antibody (rat anti-mouse IgE; BD Pharmingen) was added for 1 hr. After the plates had been washed, the enzyme avidine peroxidase (eBiosciences; Pyruvate dehydrogenase San Diego, CA) was added for 20 min. 3,3′,5,5′-tetramethylbenzidine (TMB) was used as a substrate. Absorbance was measured at 450 nm. T cells and DCs were purified from lung cell suspensions using an autoMACS separator in accordance with the manufacturer’s protocols (Miltenyi Biotec; Bergisch Gladbach, Germany). DCs and T cells were purified by positive selection using magnetic beads coupled to anti-CD11c and anti-CD3 antibodies, respectively. Purified T cells from lungs were stimulated for 18 hr with OVA (10 ng/ml) in the presence of brefeldin A (10 μg/ml). Cells were stained for cell surface markers with FITC-conjugated anti-CD4 or CD8 antibodies (BD Pharmingen). After washing, cells were fixed in 4% paraformaldehyde and permeabilized with saponin (0·1% in PBS).