Isolation and Characterization of Epidermal Cells

To generate single cell suspensions, epidermal scrapings were resuspended in complete RPMI, vortexed, pipetted to disaggregate cells, and filtered (40 μm strainer). For antigen re-expression, cells were rested overnight at 37° C with 5% CO₂ as described (15 (link)). Before staining, cells were filtered as above and counted with a Vi-cell XR (Beckman Coulter). We used validated commercial reagents: LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies), anti-mouse CD3 (17A2), CD25 (PC61.5), granzyme B (GzA-3G8.5), Lamp-1 (eBio1D4B), NK1.1 (PK136), IL-17A (XMG1.2), IL-22 (1H8PWSR) (Ebioscience), anti-mouse γδ TCR (GL3), CXCR3 (CXCR3-173), CD69 (H1.2F3), Vγ5 (536), Vγ4 (UC3-10A6), Vγ1 (2.11) (Biolegend), anti-mouse CD45 (30-F11) (BD Biosciences), on Becton-Dickinson LSR II hardware using FACSDiva software. For cytokine staining, cells were stimulated with Leukocyte Activation Cocktail with Golgiplug (BD Biosciences) for 5 h, surface stained, fixed and permeabilized with Foxp3/transcription factor buffer (Ebioscience), then stained.