Skin and peritoneal cavity cell preparations

To isolate skin cells from naïve and HDM-applied mice, shaved dorsal skin samples were minced by using scissors and incubated for 60 min in collagenase II (200 U/ml; Worthington Biochemical, Lakewood, NJ) in RPMI 1640 medium containing deoxyribonuclease I (50 U/ml; Worthington Biochemical) and 10% fetal bovine serum (FBS). Additional dissociation and homogenization were performed by using a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Cell preparations were filtered through a 55-μm nylon mesh to obtain single-cell suspensions. Peritoneal cavity cells were harvested from naïve mice and then filtered through a 55-μm nylon mesh to obtain single-cell suspensions.