Cutaneous Cell Isolation and Characterization

Human samples were obtained in accordance with a favorable ethical opinion from Newcastle, Singapore SingHealth, and National Health Care Group Research Ethics Committees. Normal skin was obtained from mammoplasty and breast reconstruction surgery and digested whole (1 × 1 cm²) as previously described (Haniffa et al., 2012 (link)) to obtain single-cell suspension. Migrating cells were collected from whole skin pieces (1 × 1 cm²) cultured in RPMI with 10% FCS, and analyzed at serial time points between 0 and 72 hr. Viability was >90% by DAPI exclusion (Sigma). Where stated, 100,000 CFSE-labeled CD14⁺ blood monocytes were cultured together with 1 × 1cm² skin piece during collagenase digestion. Shave biopsies were performed on HSC transplant patients with a DermaBlade^® (Shuco). Whole single-cell suspensions were then immunostained and analyzed by flow cytometry. Peripheral blood mononuclear cells were isolated by density centrifugation (Lymphoprep; GE Healthcare). Blood and dermal DC subsets, naive and bulk CD4⁺ T cells were isolated to >91% purity by fluorescence-activated cell sorting (FACS) using a FACSAriaII and FACSFusion (Becton Dickinson [BD]).