Surface staining was performed using fluorescein-isothiocyanate-labeled anti-ST2 (clone DJ8), allophycocyanin-labeled anti-c-kit (clone 2B8), anti-CD4 (clone RM4-5), anti-CD11b (clone M1/70), and phycoerythrin-labeled Enzalutamide chemical structure anti-Fc��RI (clone MAR-1), anti-CD3 (clone 145-2C11), anti-Ly6G (clone 1A8), and anti-F4/80 (clone BM8). Fluorochrome-conjugated antibodies were purchased from MD Biosciences (Z��rich, Switzerland), eBioscience (Vienna, Austria), or BD Biosciences (Erembodegem, Belgium). Cell acquisition was performed using a FACSCalibur flow cytometry system, and analyses were done with CellQuest Pro software 4.0 (BD Biosciences). Bone-Marrow-Derived Mast Cells Bone-marrow-derived mast cells (BMMCs) were isolated according to a previously published protocol.

21 Briefly, BMMCs were obtained by culturing femoral bone marrow cells from WT and Il1rl1?/? mice in Iscove’s modified Dulbecco’s medium (Lonza) containing 10% FBS, 100 IU/mL penicillin, 100 ��g/mL streptomycin, 250 ng/mL amphotericin B (Gibco; Life Technologies, Paisley, UK), and 5 �� 10?5 mol/L ��-mercaptoethanol. Nonadherent cells were cultured for 6 to 12 weeks in the presence of IL-3 (5 ng/mL) and stem cell factor (SCF) (50 ng/mL; Invitrogen-Life Technologies, Paisley, UK). Thereafter, cell purity was controlled by flow cytometry, revealing >90% of c-kithigh Fc��RIhigh cells, considered as differentiated mast cells. For in vitro experiments, 106 BMMCs/well were cultured for 24 hours with lipopolysaccharide (LPS) (1 ��g/mL; E. coli serotype 055:B5; Sigma-Aldrich) or with mouse rIL-33 (10 ng/mL; R&D Systems, Minneapolis, MN).

Tryptase activity in BMMC culture supernatants and in mouse sera was assayed with a mast cell degranulation assay kit (Merck Millipore, Darmstadt, Germany) according to the manufacturer’s protocol, in which calcium ionophore was used as an inducer of degranulation and protamine as an inhibitor of it. Pancreas Cell Preparation Acinar cells were isolated as described previously,22 with slight modifications. In overnight-fasted WT mice not receiving any treatment, pancreata were dissected free of mesenteric fat, minced in Hank’s buffered saline solution with 5 mmol/L EDTA solution at 37��C, and centrifuged for 2 minutes at 500 �� g twice to eliminate fat. The tissues were rinsed in Waymouth’s medium (Invitrogen-Life Technologies) containing penicillin and streptomycin (0.

1 mg/mL; Lonza), 20% FBS, and 0.01% soybean trypsin inhibitor (Sigma-Aldrich) and were digested by means of two cycles of shaking at 25 cycles/minute in a solution of 6.25 U/mL collagenase in Hank’s buffered saline solution containing 0.2% bovine serum albumin at 37��C. Nondigested tissue was eliminated by 70-��m pore filtration, and isolated cells were washed twice in complete Waymouth’s GSK-3 medium.