Primary cell culture
Primary cells were obtained by biopsy of the ear skin of calves. The ear
skin was chopped into small pieces with a sterile scalpel and then
washed several times and incubated at 38.5 °C for 4–18 h in Hank’s
Balanced Salt Solution (Gibco, Cat. no. 14175095) supplemented with
collagenase (Collagenase type I, Gibco, Cat. no. 17–100–017). The
following day the dispersed cells were washed several times in DMEM
(Gibco, Cat. no. 21068028) and cultured in DMEM supplemented with 10%
fetal calf serum (Gibco, Cat. no. GIB-11150–059), 1%
penicillin/streptomycin (Gibco, Cat. no. 15140148), 1% non-essential
amino acids (Gibco, Cat. no. 11140050), and 100 mM β-mercaptoethanol
(Sigma–Aldrich, Cat. no. M3418).