[38] Detection of cell surface components using fluorescent microspheres

This chapter describes the detection of cell surface components using fluorescent microspheres. Fluorescent microspheres offer the potential for enhanced sensitivity because they can be manufactured to possess the fluorescence equivalent of several thousand fluorescein molecules per sphere. Streptavidin-mediated coating of biotinylated fluorescent microspheres (BFM) with either biotinylated antibody or another biotinylated cell surface probe increases the number of probe molecules immobilized, thus increasing their effective concentration and the probability of binding to the surface determinant. Streptavidin coating of BFM involves sonicating the BFM stock for 10 minutes. The streptavidin–BFM complexes are separated from unbound streptavidin by gel filtration through Sepharose 4B. Glass columns fitted with porous polyethylene disks will permit the passage of particles of up to 30 μm. The method of coating streptavidin-BFM with biotinylated probe is analogous to the procedure for coating BFM with streptavidin. The biotinylated probe is incubated with streptavidin–BFM with gentle rocking for 30 minutes at room temperature. The shelf life of each unique BFM probe must be determined empirically by the investigator.