A new standard fluorescence microsphere for quantitative flow cytometry.

A standard fluorescence microsphere has been prepared for quantitative flow cytometry. A uniformly sized (6.53 +/- 0.34 micron in diameter) and perfectly spherical plastic microsphere, Micropearl-FT, was covalently conjugated with fluorescein isothiocyanate (FITC). The characteristic of this microsphere to lyse completely in strong alkaline solutions made it possible to determine the amount of bound FITC easily and reproducibly. The amount of bound FITC depended on the concentration of FITC and the incubation time at pH 9.5 and 37 degrees C. In flow cytometry, the fluorescence intensity of the FITC-conjugated microsphere (FCMS) showed linear dependence on the amount of bound FITC. The fluorescence intensity of FCMS was stable for at least 3 months even in aqueous solutions. Using FCMS, an Epics V was calibrated and the minimum measurable fluorescence intensity was examined. From analysis using this system, the number of FITC-conjugated OKT3 molecules bound to human T lymphocytes was shown to average 4.4 X 10(4) per cell at saturation.