UNIT 6.21 Detection of Cytokine Receptors by Flow Cytometry

This unit describes three protocols for detecting cytokine receptors on human peripheral blood mononuclear cells or murine splenic lymphocytes via immunofluorescence labeling. The three protocols have in common the use of phycoerythrin as a highly effective fluorescent dye, multiple-layer staining to increase sensitivity, and selection of reagents for maximum sensitivity. The first procedure uses monoclonal antibody (MAb) against receptor protein, detected with biotinylated anti-Ig (directed against the species in which the MAb is made), which in turn is stained with phycoerythrin-streptavidin (PE-SA). The second procedure uses biotinylated cytokines in the first step, followed by PE-SA. Finally, a third procedure describes multiparameter analysis involving second and third fluorophores introduced as direct antibody conjugates, which is used to study subset expression of receptors. Instructions are also provided for titrating MAbs and cytokines, as well as how to evaluate detection reagents such as anti-Ig conjugates and fluorophores.