Isolation, characterization and in vitro cultivation of subfractions of neonatal mouse keratinocytes: effects of phorbol esters

Abstract

Using discontinuous Percoll density gradient centrifugation, keratinocytes from neonatal mouse skin were separated into four fractions. The fractions were characterized by light microscopy, by indirect immunofluorescence using lectins ( Bandeira simplicifolia and Ulex europaeus ) or an antibody against buUous pemphigoid antigen, and by electrophoretic analysis of the keratin polypeptide patterns. Two fractions, Fl and F2, were identified as being derived from suprabasal layers and two fractions, F3 and F4, as originating from the basal cell layer. F3 and F4 cells could be cultivated on plastic substratum in the presence of 4 x minimum essential medium, 10% fetal calf serum at 34°C with plating efficiencies of 80–85%. DNA labeling of cultures of F3 and F4 cells indicated that basal cells in fractions F3 and F4 exhibited different growth behaviour in culture. Confluent cultures of F3 and F4 cells responded to phorbol esters by increased DNA synthesis. This result contradicts the in vivo situation where neonatal mouse skin is resistant to the irritant and mitogenic effects of phorbol esters. The mitogenic effect of 12- O -tetra-decanoylphorbol-13-acetate on F3 cells is not mediated by endogenous prostaglandin formation.