Leukocyte isolation from skin

Ears from infected mice were removed and dorsal and ventral sides of the ear pinna were separated and incubated in 1 mL HBSS (Gibco) containing CaCl₂ and MgCl₂ supplemented with 125 U/ml collagenase II (Invitrogen) and DNAse-I (Sigma) at 37° C for 1 hour. Whole tissue suspensions were then generated by forcing the digested skin through a wire mesh screen. Leukocytes were then purified by resuspending the cells in 10 ml of 35% (v/v) Percoll (GE Healthcare) in HBSS in 50 ml conical tubes followed by centrifugation for 10 minutes at room temperature.