Cell preparation and flow cytometry

Collected skin biopsies comprising the four excisional wounds were cut into small pieces using scissors and scalpel before overnight treatment in a 2.5 U/ml solution of dispase diluted in complete RPMI at 4°C. Then, skin tissues were incubated in an enzymatic solution composed of 50 µg/mL of Liberase TM Research Grade (Roche Diagnostics, Basel, Switzerland), 25 ng/ml of collagenase I and 100 µg/mL of DNase I (Sigma-Aldrich, St. Louis, MO) for 4 hours at 37°C before filtration through a 100 µm cell strainer followed by a second filtration through a 40 µm cell strainer and two washes in complete RPMI by centrifugation at 1700 rpm for 10 min. After the last centrifugation, cell pellets were resuspended with 250 µl of StemPro Accutase solution (Gibco) for 2 min at 37°C. After washing, cells were resuspended in complete RPMI and stimulated for 4 hours in the presence of phorbol myristate acetate (PMA, 50 ng/ml), ionomycin (750 ng/ml) and Golgi Plug (1 µl for/10⁶ cells; BD Biosciences). Before extracellular staining, cells were incubated with Fc Block (BD Biosciences) for 10 minutes and then for 15 minutes at 4°C with the following antibodies: V500-conjugated anti-CD45, BV421-conjugated anti-CD3ϵ, FITC-conjugated anti-TCRγδ (all from BD Biosciences) and Zombie NIR for cell viability (BioLegend, San Diego,CA). For intracellular staining, cells were permeabilized with the Cytofix/Cytoperm kit and labelled for 15 min at 4°C with PE-conjugated anti-IL-17A (BD Biosciences) and PercP-eFluor710-conjugated anti-IL-22 (eBioscience) antibodies. Data were collected on a FACS Verse instrument (BD Bioscience) and analyzed using FlowJo software.