Cytofluorometric quantification of IgE and IgE receptors on individual mast cells
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Published on: December 1, 2014
X J Chen1, S Juliusson, F Aldenborg
1Department of Pathology, Sahlgrenska Hospital, University of Göteborg, Sweden.
Insights
A new cytofluorometric method quantifies immunoglobulin E (IgE) and IgE receptors on mast cells. This technique reveals significant variability in IgE content among mast cells, even in healthy rats.
Area of Science:
- Immunology
- Cell Biology
- Biophysics
Background:
- Understanding the mast cell-IgE system is crucial for immunological research.
- Quantifying cell-bound IgE and IgE receptors on mast cells requires precise methodologies.
Purpose of the Study:
- To develop and validate a cytofluorometric method for measuring cell-bound IgE and IgE receptors on individual mast cells.
- To investigate IgE content and receptor saturation in rat peritoneal mast cells under normal conditions.
Main Methods:
- Exploration of a cytofluorometric method using rat peritoneal mast cells and rat basophilic leukaemia (RBL-1) cells.
- Systematic optimization of fixation, incubation time, temperature, and antibody concentrations.
- Standardization using polystyrene beads and fluorescent uranyl glass.
- Direct labeling with FITC-conjugated goat anti-rat IgE(Fc) for quantitative single-cell analysis.
Main Results:
- Established optimal conditions for quantitative IgE measurement at the single-cell level.
- Demonstrated a linear relationship between fluorescence intensity and IgE concentration in RBL-1 cells.
- Quantified IgE content and receptor saturation in peritoneal mast cells of pathogen-free Sprague-Dawley rats.
- Observed significant variability in IgE content, with up to 30% of mast cells having little or no IgE, while IgE receptors were rarely absent.
- Found that 60-70% of available IgE receptors were occupied by IgE in normal rats.
Conclusions:
- The developed cytofluorometric method enables accurate quantification of IgE and IgE receptors on individual mast cells.
- Mast cell IgE content exhibits considerable heterogeneity, even in healthy individuals.
- IgE receptor expression is generally consistent, but IgE binding varies significantly, suggesting dynamic regulation of the mast cell-IgE system.
Abstract:
Information about the IgE receptor and IgE content of mast cells under different conditions in vivo is essential for further understanding the functions of the mast cell-IgE system. A cytofluorometric method for measuring cell-bound IgE on individual mast cells was therefore explored 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 variables such as fixation, incubation time, temperature and concentrations of antibody on the fluorescence emission of the mast cells. Optimum conditions were selected for the quantitative measurement of IgE at the 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 measurement procedure. A linear relationship between fluorescence intensity and IgE concentration was obtained by fluorescence measurements on RBL-1 cells incubated in rat IgE. In the case of rat peritoneal mast cells it was possible to saturate the IgE receptors by incubating the mast cells in rat IgE. By measuring the mast cells before and after IgE incubation, the relative content of IgE, the relative number of IgE receptors and the degree of receptor saturation could be estimated. In this way we measured the IgE content of peritoneal mast cells of Sprague-Dawley rats maintained under pathogen-free conditions. The distribution of IgE content in the mast-cell populations was extremely variable. Up to 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 average, 60-70% of the receptors available for binding were occupied by IgE in these normal rats.
