CYTOFLUOROMETRIC QUANTIFICATION OF IGE AND IGE RECEPTORS ON INDIVIDUAL MAST-CELLS

Information about the IgE receptor and IgE content of mast cells under different conditions in vivo is essential for further understanding t he functions of the mast cell-IgE system. A cytofluorometric method fo r measuring cell-bound IgE on individual mast cells was therefore expl ored using peritoneal mast cells of Sprague-Dawley rats infected with the nematode N. brasiliensis and rat basophilic leukaemia cells (RBL-1 ) as experimental models. We systematically studied the effects of var iables such as fixation, incubation time, temperature and concentratio ns of antibody on the fluorescence emission of the mast cells. Optimum conditions were selected for the quantitative measurement of IgE at t he single-cell level using direct labelling with FITC-conjugated goat anti-rat IgE(Fc). Polystyrene beads with a defined fluorophor content and a fluorescent uranyl glass were used to standardise the measuremen t procedure. a linear relationship between fluorescence intensity and IgE concentration was obtained by fluorescence measurements on RBL-1 c ells incubated in rat IgE. In the case of rat peritoneal mast cells it was possible to saturate the IgE receptors by incubating the mast cel ls in rat IgE. By measuring the mast cells before and after IgE incuba tion, the relative content of IgE, the relative number of IgE receptor s and the degree of receptor saturation could be estimated. In this wa y we measured the IgE content of peritoneal mast cells of Sprague-Dawl ey rats maintained under pathogen-free conditions. The distribution of IgE content in the mast-cell populations was extremely variable. Up t o 30% of the mast cells in individual populations contained little or no IgE, but very few if any of the cells lacked IgE receptors. On aver age, 60-70% of the receptors available for binding were occupied by Ig E in these normal rats.