Open AccessCell kinetic characterization of the epidermal growth factor dependent BALB/MK line using flow cytometric analysis of DNA content and iododeoxyuridine incorporation
Author(s) -
Van Hooijdonk C. A. E. M.,
Van Erp P. E. J.,
Freund R. L.,
De Jongh G. J.,
Schalkwijk J.,
Van De Kerkhof P. C. M.,
Mier P. D.
Publication year - 1993
Publication title -
cell proliferation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.647
H-Index - 74
eISSN - 1365-2184
pISSN - 0960-7722
DOI - 10.1111/j.1365-2184.1993.tb00034.x
Subject(s) - trypsinization , cell cycle , flow cytometry , population , epidermal growth factor , biology , fetal bovine serum , microbiology and biotechnology , cell culture , dna synthesis , cell growth , cell , andrology , immunology , chemistry , biochemistry , trypsin , medicine , dna , genetics , environmental health , enzyme
Epidermal hyperproliferation (psoriasis, wound repair) is the result of quiescent (G 0 ) keratinocytes being recruited into the cell cycle. A detailed characterization of the cell cycle kinetic parameters of the mouse keratinocyte line (Balb/MK) has been carried out with the aid of bivariate iododeoxyuridine (IdUrd) and DNA analysis using flow cytometry, in order to establish whether it might provide a useful model for the study of the biochemical events controlling recruitment into the cell cycle. Balb/MK keratinocytes were cultured using low Ca 2+ Dulbecco's modified Eagle's medium/F12 in the presence of 10% dialysed fetal bovine serum and 4 ng/ml epidermal growth factor (EGF). IdUrd labelling followed by flow cytometric analysis of trypsinized cells showed that about 95% of the population were actively cycling, with a cell cycle time of around 14 h and no significant contact inhibition. Omission of serum and EGF followed by IdUrd pulse‐labelling indicated that the cells progressively withdrew from the cycle and, after 16h, less than 10% incorporated IdUrd. Subsequent restimulation with serum resulted in a synchronized cohort of cells being recruited. Entry into the S phase of the cell cycle (IdUrd incorporation) started at 8 h and was maximal between 12 h and 16 h after stimulation. Restimulation with EGF alone reached a growth fraction of 87% after 24 h continuous labelling compared with 97% using serum together with EGF. Epidermal growth factor already showed a significant stimulation at 10 pg/ml compared with the controls. There is a broad plateau centred on 5 ng/ml, followed by a slight decline above this level. We conclude that the use of a cell line with defined cell cycle kinetic parameters which can be switched between the quiescent and cycling states in a fully defined medium will provide an ideal model for biochemical studies of the relevant signal transduction pathways in keratinocytes.