Isolation, characterization, and in vitro cultivation of keratinocyte subfractions from adult NMRI mouse epidermis: epidermal target cells for phorbol esters.

Adult dorsal mouse epidermis (strain NMRI) was separated from dermis in thin-split sections by cold trypsinization. From the isolated keratinocytes four cell fractions (F1-F4) were obtained using discontinuous Percoll density gradient centrifugation. The fractions were characterized by light microscopy, by indirect immunofluorescence using specific lectins (Bandeirea simplicifolia and Ulex europaeus) and an antibody against the spinous 67-kDa keratin polypeptides, and by electrophoretic analysis of the keratin polypeptide patterns. The heavy fractions, F3 and F4, were identified as being derived from the basal cell layer, whereas the light fractions, F1 and F2, consisted mainly of suprabasal cells. The basal cells (F3 and F4) could be cultivated on plastic substratum coated with rat-tail collagen (4 X MEM, 10% FCS at 34 degrees C; plating efficiency 70-85%). Labeling of DNA with [3H]thymidine indicated that during the first 5 days of cultivation, basal cells ran through two cell cycles, after which the proliferative activity ceased due to terminal differentiation. The addition of the tumor promoter TPA led to a stimulation of DNA synthesis in confluent cultures of both F3 and F4 cells.