Journal of Dermatological Science, 1 (1990) 47- 56 Elsevier DESC 00011 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Reactivity of monoclonal anti-human skin basal cell antibody to cultured keratinocytes in serum-free medium Fukumi Furukawa , 1,3 David A. Norris3, Mari Kashihara-Sawami 1,3, Martin B. Lyons 3, Masamichi Ueda2 and Sadao Imamura l ‘Department of Dermatology, Faculty of M edicine. 2Department of Pathology. Institute for Virus Research, Kyoto University, Kyoto, Japan, and 3Department of Dermatology, Universitv of Colorado School of Medicine, and Denver Veterans Administration Medical Center, Denver, Colorado, U.S.A. (Received 10 July 1989; accepted 24 October 1989) Key words: Cultured keratinocytes; Calcium concentration; Basal cell antigen zyxwvutsrqponmlkjihgfedcba Abstract The reactivity of the monoclonal anti-human skin basal cell antibody (3B4-6) to cultured keratinocytes was examined by immunofluorescence techniques in order to verify the change in distribution ofbasal cell antigens during the in vitro differentiation of keratinocytes cultured in serum-free medium. The basal cells’ antigen(s) determinants recognized by 3B4-6 were present in the cytoplasm of the cells at low concentration of Ca’ + (0.03 mM). High concentrations of Ca2 + (0.1 mM x 0.3 mM) induced the reactivity on the cell surface. This result was confirmed by flow cytometry analysis. The staining pattern was different from that of anti-involucrin antibody in 0.3 mM Ca’+ culture medium. This MoAb might be a useful tool for monitoring the degree of differentiation of cultured keratinocytes. Introduction Skin basal cells are essential for cell growth and cell differentiation in the stratified epithelium of the skin tissue. Skin basal cells are con- ventionally recognized by the reactivity to the cells of the sera of patients with pemphigoid [ 11, and Correspondence to: Fukumi Furukawa, M.D., Department of Dermatology, Faculty of Medicine, Kyoto University, Shogoin, Sakyo, Kyoto 606 Japan. Abbreviations: MoAb: monoclonal antibody; FCS: fetal calf serum; HK: human keratinocyte; AO: acridine orange; EB: ethidium bromide; PI: propidium iodide; RT: room tempera- ture; PBS: phosphate buffered saline; WBPE: whole bovine pituitary extract. 0923-181 l/90/$03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division) of monoclonal antibodies (MoAb) against skin basal cells. These MoAb have been developed by several investigators [2-51. Recently, we reported the establishment of a MoAb against skin basal cells [ 51. This MoAb is characterized by a specific reactivity to skin basal cells including melano- cytes, by the lack of reactivity to the basement membrane zone, and by a cross-reactivity with the vascular components such as endothelial cells and pericytes of the capillaries, capillary endothe- lium of the renal glomeruli, hepatic sinusoids and alveolar walls of the lung [6]. Cultured keratino- cytes are now commonly used for modem investi- gation in dermatology. One major area of interest in cutaneous cell biology is the study of differen-