Isolation of skin-infiltrating leukocytes in murine psoriasis

Skin-infiltrating leukocytes were isolated from the back skin or left ear skin of IMQ-induces psoriasis mice (28 (link)). After 7 days of IMQ application, the 1 cm² area from the mouse back skin and left ear were cut off, removed with remaining IMQ cream by HBSS (Gibco) wash and separated with epidermis and dermis using curved forceps. Separated epidermis and dermis were digested with Dispase II solution (5 mg/ml, Sigma Aldrich) at 37°C. After first digestion, the dermis was cut quickly into small pieces (< 0.5 mm) with curved scissors in Dermis Dissociation buffer (DMEM containing 1 mg/ml of collagenase P and 100 μg/ml of DNaseI (Hyclone, Roche) and incubated at 37°C for 1 hr. The fully digested dermal suspension was filtered through a 40-μm cell strainer (Falcon) and rinsed with DMEM medium containing 10% FBS (Hyclone). Isolated dermal-infiltrating leukocytes were collected by centrifugation. The epidermis, after first digestion, was cut quickly into small pieces (< 2 mm) with curved scissors and transferred to the 0.05% TE (Trypsin-EDTA, Hyclone) buffer and incubated at 37°C for 5 min. After incubation, trypsin neutralizing solution (5% FBS diluted with HBSS) was added to the fully digested epidermis, and the epidermal suspension was filtered through a 40-μm cell strainer (Falcon). Isolated epidermal-infiltrating leukocytes were collected by centrifugation.