Dispase 2 solution

The epidermis was removed from the dorsal skin of newborn mice overnight at 4 °C with 0.2% Dispase II solution (Sigma-Aldrich, St Louis, MO, USA). The next day, the dermis was digested with 0.25% collagenase IV solution (Sigma-Aldrich) at 37 °C for 1 h. After filtration, centrifugation and resuspension, MDFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco Life Technologies) and 1% penicillin/streptomycin (Gibco Life Technologies) at 37 °C in 5% CO2. Passage 3-5 MDFs were used for subsequent experiments.

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.

Primary human epidermal keratinocytes (PHEKs) and melanocytes (PHEMs) were isolated from equal size (1 cm × 1 cm in square) of human young adult foreskin samples obtained in the surgery of circumcision. For culture of PHEKs, the foreskin sample was initially immersed in 10 ml of 0.2% Dispase II solution (Sigma, St. Louis, MO, USA) at 4 °C for 48 h and diced in pieces, followed by incubation in 0.05% Trypsin-EDTA solution for 15 min. The pelleted cells were obtained by a centrifugation at 1,300 rpm for 5 min, seeded in a fibronectin/collagen (AthenaES, Baltimore, MD, USA) coated flask and cultured in EpiLife^® keratinocyte medium at 37 °C in 5% CO₂. For primary culture of PHEMs, the epidermal cell cultured in the EpiLife^® medium were transferred to melanocyte culture medium after the epidermal primary culture and incubated at 37 °C in 5% CO₂. Highly selected culture of PHEMs was then obtained after passage 2.

The normal human skin tissues or neonatal mouse dorsal skin were placed in a 0.2% Dispase II solution (Sigma‐Aldrich, St Louis, MO, USA) overnight at 4 °C. The next day, the dermal skin was digested with 0.25% collagenase IV solution (Sigma‐Aldrich) at 37 °C for 2 h. After filtration, centrifugation, and resuspension, the human dermal fibroblasts (HDFs) or mouse dermal fibroblasts (MDFs) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco Life Technologies) and 1% penicillin/streptomycin (Gibco Life Technologies) at 37 °C and 5% CO₂. Passage 3–5 cells were used for experiments.

Low methoxy (LM) pectin was purchased from CP Kelco, UK; collagen (PureCol EZ Gel, Advanced BioMatrix), CaCl₂·2H₂O, DMEM, HEPES, adipogenic media, dispase II solution, trypsin, penicillin/streptomycin, and paraffin were all purchased from Sigma-Aldrich, UK; keratinocyte growth medium was purchased from KGM, Lonza, UK; and calcein AM (acetoxymethyl ester) and propidium iodide were purchased from Fisher Scientific, UK.

Confluent monolayers were subjected to treatment as indicated in the respective experiment. Afterwards, cells were detached from the well bottom by application of 200 μl Dispase-II solution (Sigma-Aldrich) for 20 min. Following successful detachment, 350 μl Hank's Balanced Salt Solution (HBSS) was used to substitute the enzyme. Defined mechanical shear stress was applied with an electrical pipette (Finnpipette Novus, ThermoFisher, Waltham, USA). Cell-sheet fragments correlate with loss of adhesion (19 (link)) and were counted under a binocular microscope (Stemi 508, Zeiss, Jena, Germany). Pictures were taken with a Canon EOS 600D camera (Krefeld, Germany).