Isolation of Epidermal Mononuclear Cells

Abdominal skin was collected immediately after surgery and processed as previously described [35 (link)]. Briefly, skin pieces were removed from underlying tissue using a skin graft knife (Swann-Morton, UK), and the resulting skin grafts were passed through a skin graft mesher (Zimmer Biomet, USA). To facilitate the mechanical separation of the epidermis from underlying dermis, the meshed skin pieces were placed in RPMI 1640 (Lonza) containing 0.14 U/mL dispase (Worthington Industries, USA) and 50 μg/mL gentamicin, and rotated at 4°C overnight. The skin was then washed in PBS and epidermis was separated from dermis using fine forceps. To liberate epidermal cells, epidermal tissue was incubated with RPMI 1640 containing 200 U/mL collagenase Type IV (Worthington) at 37°C for 120 min. A tea strainer was used to separate the cells from undigested tissue, and then the supernatants were passed through a 100 μm cell strainer (Greiner Bio-One, Germany). The epidermal cells were pelleted, washed twice with cold PBS and resuspended in RPMI 1640 for centrifugation on a Ficoll-Paque PLUS (GE Healthcare, USA) density gradient at 450 x g for 20 min to harvest the epidermal MNPs. For Caspase 3 experiments, a Dead Cell Removal kit (StemCell Technologies, Canada), was used as per manufacturer’s directions to remove dead cells prior to culture.