Detection of cytokine receptors by high-sensitivity immunofluorescence/flow cytometry.
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.
Zola
H
H
Flinders Medical Centre, Bedford Park, Australia.
Flego
L
L
Sheldon
A
A
D000818:Animals; D016207:Cytokines / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D011971:Receptors, Immunologic / Q000032:analysis*
D016207:Cytokines; D011971:Receptors, Immunologic