Isolation of Epidermal and Dermal Cells

Epidermal and dermal cell suspensions were prepared as before^{4 (link)} with slight modification. Briefly, shaved whole trunk skin was harvested from appropriate animals and, after removal of subcutaneous tissues with forceps, was floated with epidermal side up onto 10 mL of Trypsin-EDTA solution containing 5 mL of 0.25% Trypsin (Nacalai tesque) and 5 mL of 0.05% Trypsin-0.53 mM EDTA • 4Na (Nacalai tesque) at 37°C for less than 30 minutes in 10 cm dish. Epidermis and dermis were separated manually with forceps. Dermis was cut into small chips manually with scissors and further incubated in 4 mL of RPMI containing 0.03% Liberase TL Research grade (Roche Applied Science) and 200 U/mL of DNase (Wako Pure Chemical Industries) for 60 minutes at 37°C shaking with rotation at 200 rpm (Bioshaker, Taitec). Epidermal and dermal cells were then suspended in 5% FCS in PBS, washed and filtered through Cell strainer (BD Falcon).