Green- and red-fluorescent nanospheres for the detection of cell surface receptors by flow cytometry

Fluorescent probes serve as sensitive tools for obtaining structural and functional information in cellular systems. In spite of the high sensitivity provided by fluorescent reagents, cell surface receptors expressed in low numbers often escape detection with commonly used fluorescent probes. R-Phycoerythrin (R-PE), a molecule with a very high quantum yield, is often the reagent of choice for the detection of such low abundance events. We have developed streptavidin conjugates of two highly fluorescent 35-40 nm diameter polystyrene nanospheres, the green fluorescent FluoSpheres (Ex/Em 505/515) and red fluorescent TransFluoSpheres (Ex/Em 488/645). Like R-PE, the new reagents have peak excitations near 488 nm but differ in their emission maxima; 515 nm for the green nanospheres, 645 nm for the red nanospheres and 575 nm for R-PE. Hence the nanospheres are detected by flow cytometry in channels capable of detecting green (FL1) and red (FL3) fluorescence, while R-PE is detected in channel FL2. These nanospheres were tested for the detection of the sparsely expressed epidermal growth factor receptor (EGFR) of NIH-3T3 cells and the densely expressed EGFR of A431 cells. Results indicate that the nanosphere reagents are more sensitive than fluorescein-streptavidin and at least comparable in sensitivity to R-PE-streptavidin. The simultaneous use of these nanospheres with R-PE was also studied by concurrent staining of the CD3 and CD4 receptors in JURKAT cells. Labeling of CD4 receptors with streptavidin nanospheres and CD3 receptors with the R-PE-anti-CD3 conjugate confirmed the suitability of using the new nanospheres in combination with R-PE in multicolor flow cytometry experiments. This paper thus describes the use of alternative tools with detection sensitivity comparable to that of R-PE, but detected in different channels than R-PE, permitting their simultaneous use with R-PE.