Detection of cytokine receptors by high-sensitivity immunofluorescence/flow cytometry. 1992

H Zola, and L Flego, and A Sheldon
Flinders Medical Centre, Bedford Park, Australia.

Cytokines have profound effects on cells, and act through receptors which need only be at low concentrations (around 100 copies per cell) to transmit activation signals. The detection of such low concentrations is possible using monoclonal antibodies and fluorescence/flow cytometry, but only by using specialized techniques. The best results so far have been obtained using biotinylated second antibody followed by phycoerythrin-streptavidin, and batches of these reagents have to be carefully selected. Analysis of the fluorescence is best done using 546 nm excitation from a mercury arc lamp, but 512 nm excitation from an argon-ion laser can also be used. With appropriate alignment, instruments with 488 nm fixed-wavelength lasers can give sensitivity almost as good as the 546 nm system. Working at high sensitivity, background levels also increase, particularly for B lymphocytes. Background staining can be reduced to acceptable levels by blocking the two major mechanisms for non-specific binding. Applications of these methods to the detection of cytokine receptors on normal and malignant cells are reviewed.

UI MeSH Term Description Entries
D011971 Receptors, Immunologic Cell surface molecules on cells of the immune system that specifically bind surface molecules or messenger molecules and trigger changes in the behavior of cells. Although these receptors were first identified in the immune system, many have important functions elsewhere. Immunologic Receptors,Immunologic Receptor,Immunological Receptors,Receptor, Immunologic,Receptors, Immunological
D005434 Flow Cytometry Technique using an instrument system for making, processing, and displaying one or more measurements on individual cells obtained from a cell suspension. Cells are usually stained with one or more fluorescent dyes specific to cell components of interest, e.g., DNA, and fluorescence of each cell is measured as it rapidly transverses the excitation beam (laser or mercury arc lamp). Fluorescence provides a quantitative measure of various biochemical and biophysical properties of the cell, as well as a basis for cell sorting. Other measurable optical parameters include light absorption and light scattering, the latter being applicable to the measurement of cell size, shape, density, granularity, and stain uptake. Cytofluorometry, Flow,Cytometry, Flow,Flow Microfluorimetry,Fluorescence-Activated Cell Sorting,Microfluorometry, Flow,Cell Sorting, Fluorescence-Activated,Cell Sortings, Fluorescence-Activated,Cytofluorometries, Flow,Cytometries, Flow,Flow Cytofluorometries,Flow Cytofluorometry,Flow Cytometries,Flow Microfluorometries,Flow Microfluorometry,Fluorescence Activated Cell Sorting,Fluorescence-Activated Cell Sortings,Microfluorimetry, Flow,Microfluorometries, Flow,Sorting, Fluorescence-Activated Cell,Sortings, Fluorescence-Activated Cell
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D016207 Cytokines Non-antibody proteins secreted by inflammatory leukocytes and some non-leukocytic cells, that act as intercellular mediators. They differ from classical hormones in that they are produced by a number of tissue or cell types rather than by specialized glands. They generally act locally in a paracrine or autocrine rather than endocrine manner. Cytokine

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