Isolation of Immune Cells from Blood and Skin

Neutrophils were isolated from whole blood using Mono-Poly resolving medium (DS Pharma Biomedical), as described previously (71 (link)). We seeded neutrophils in an 8-well chamber slide (Lab-Tek) in 400 μL of serum-free medium, RPMI-1640, at a density of 10⁵ cells/well. PBMCs were isolated from whole blood using Ficoll-Paque PLUS (GE Healthcare). CD4, CD8, HLA-DR, and CD14 positive cells were isolated from PBMCs using CD4, CD8, HLA-DR, and Monocyte Isolation Kits (Miltenyi Biotec) per the manufacturer’s protocol. NHEK was purchased from Kurabo and cultured on EpiLife medium supplemented with insulin (10 mg/mL), rhEGF (epidermal growth factor, 0.1 ng/mL), hydrocortisone (0.5 mg/mL), gentamicin (50 mg/mL), amphotericin B (50 ng/ml), and bovine pituitary extract (0.4% v/v); EpiLife-KG2 medium, all from Kurabo) in a humidified atmosphere with 5% CO₂ at 37°C. Monocytes in blister fluids were isolated from whole blister fluids using a Monocyte Isolation Kit (Miltenyi Biotec) per the manufacturer’s protocol.