Materials and Methods Animals This study was approved by the Animal Ethics Committee in Gothenburg, Sweden. The permit number is Dnr442–2008 data 2008-12-12. Male C57BL/6 mice, 5 to 6 weeks old, were purchased from Taconic (Ry, Denmark). All mice were kept under standard animal housing conditions and provided with food and water ad libitum. Allergen sensitisation and allergen exposure Mice were sensitised twice, with an interval of five days, by the intraperitoneal (i.p.) injection of 0.5 ml of 8 mg chicken ovalbumin (OVA)(Sigma-AldrichH, St Louis, MO, USA) bound to 4 mg aluminium hydroxide (Sigma-AldrichH) in phosphate buffered saline (PBS). Eight days after the second sensitisation, the mice were divided in two groups. Both groups were briefly anesthetised using isoflurane (Baxter, Deerfield, IL, USA). The exposure group received an intranasal (i.n.) administration of 100 mg OVA in 25 ml of PBS on five consecutive days, while the control group received only PBS (Fig. 1A). Evaluation of newly produced inflammatory cells BrdU (5-bromo-29-deoxyuridine) is a thymidine analogue that is incorporated into the DNA during the S-phase of the cell cycle by replacing thymidine. All mice were given 4 mg of BrdU (BrdU Flow Kits, BD PharmingenTM, San Diego, CA) to label newly produced cells. BrdU was given at a dose of 0.8 mg in 0.2 ml of PBS by i.p. injection once a day just after allergen exposure, on days 1 to 5 of allergen exposure. Sample collection and processing Samples were collected 24 hrs after the final OVA exposure. The animals were deeply anesthetised with a mixture of xylazin (130 mg/kg, RompunH, Bayer, Germany) and ketamine (670 mg/ kg, KetalarH, Parke-Davis, England). Firstly, animals were sacrificed by puncturing the right heart ventricle and blood was collected. Secondly, the mice were tracheotomised and broncho- alveolar lavage (BAL) was performed by instilling 0.25 ml PBS through the tracheal cannula, followed by gentle aspiration and a second lavage with 0.20 ml PBS. An additional 1 ml of PBS was used to wash away airway lumen inflammatory cells, before the Lung Expansion of Th1, Th2, Th17 and Tregs lungs were perfused and removed to harvest parenchymal inflammatory lung cells. The left lobe was filled with 1 ml of Tissue-TekH (Sakura Finetek Europe B.V., 2382 AT, Zoeter- woude, NL) and PBS containing 20% sucrose (Sigma-AldrichH) and immediately frozen in liquid nitrogen. The apical lobe was stored at 280uC and another three lobes, without any connective tissue, were stored on ice in Hanks balanced salt solution (HBSS) (Sigma-AldrichH) before use. Preparation of lung single-cell suspensions The right lung lobes were weighed and rinsed in a Petri dish before being transferred to a gentleMACSTM C Tube (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) containing 5.0 ml of HBSS supplemented with 10% fatal calf serum (FCS) (Sigma- AldrichH), 100 ml Collagenase D solution (final concentration 2 mg/ml) and 20 ml DNase I solution (final concentration 80 U/ ml). The mouse lung was dissociated using a gentleMACS Dissociator (Miltenyi Biotec) according to the manufacturer’s instructions. Lung cell pellets were collected, washed in PBS supplemented with 10% FCS and the cells saved for FACS analysis. The total number of cells was determined using standard haematological procedures. Flow cytometry and gating strategy Flow cytometric analysis of viable leucocytes, eosinophils and T cells. Cells were pre-treated with 2% mouse serum (DAKO) for 15 min to prevent unspecific binding and thereafter stained with the following antibodies; Fluorescein (FITC) labelled anti-CD45 (clone 30-F11, BD Biosciences), Peridinin Chlorophyll Protein Complex (PerCP) labelled anti-CD3e (clone 145-2C11), Phycoerythrin (PE) labelled anti-CD4 (clone H129.19, BD Biosciences) and 7-aminoactinomycin (7-AAD) (BrdU Flow Kits, BD PharmingenTM, San Diego, CA) or a matching isotype control antibody. The cells were incubated for 30 min at 4uC with the antibodies or isotype controls, followed by two washes with washing buffer (PBS +10% FCS) and fixed with Fix/ Perm solution (Foxp3 Staining Buffer Set, eBioscienceTM) overnight at 4uC. The remainder of the protocol is as described for the intracellular staining below, with the exception of the antibody addition. Gating was first set on intact cells based on forward and side scatter characteristics. Viable leucocytes were identified as 7-AAD-CD45+ cells in a gated population based on Figure 1. Establishment of allergic airway inflammation. A) Schedule of OVA sensitisation (i.p.), OVA exposure (25 ml intranasal instillation: i.n.), BrdU administration (1 mg i.p.) and tissue sampling in this model of allergic airway inflammation. All animals were sensitised to allergen, but control animals were exposed to PBS instead of OVA. B) Number of lung tissue CD45+ CCR3+ granulocytes (i.e. eosinophils) per gram of tissue after OVA/PBS or OVA/OVA exposure, Data is shown as mean 6 SEM, n = 5–8 mice/group, ***, p,0.001. doi:10.1371/journal.pone.0019889.g001 PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e19889