A technique for the study of endocytosis in human oral epithelial cells

Fluorescently labelled latex microspheres (0.01, 0.1 and 1.0 mu m dia.) were used to establish whether oral epithelial cells could exhibit an endocytic function. Oral mucosa biopsies were incubated in organ culture at 37 degrees C for 20 h with one of the three sizes of fluorescent microspheres in the medium. Tissue pieces were then disaggregated and cell suspensions analysed for cell content and viability. Evidence of endocytosis was sought by using fluorescence-activated cell scanning (FACS) and confocal microscopy to study the epithelial cell suspensions for internalization of the microspheres. Confirmation that the microspheres had been internalized and were not merely attached to the cell exterior was shown by using trypan blue quenching to extinguish extracellular fluorescence, allowing analysis of only intracellular fluorescent microspheres. Both FACS and confocal microscopy confirmed uptake of 0.01 and 0.1 I tm dia. microspheres but not 1.0 mu m. Endocytosis was quantitated using FACS and a dose-dependent relation between the concentration of spheres in the incubation medium and uptake was found. Internalization of microspheres of <1.0 mu m dia. and the dose-dependent uptake support a fluid-phase constitutive endocytic process. (C) 1999 Elsevier Science Ltd. All rights reserved.