Development of Multicolor Flow Cytometry Calibration Standards: Assignment of Equivalent Reference Fluorophores (ERF)...

A procedure is described for assigning the number of equivalent reference fluorophores (ERF) values to microspheres labeled with a fluorophore designed to produce a fluorescence response in a given fluorescence channel of a multicolor flow cytometer. A fluorimeter was calibrated by a series of solutions of the reference fluorophores. The fluorimeter was used to obtain the microsphere fluorescence intensity, and a multicolor flow cytometer was used to obtain the microsphere concentration. The microsphere fluorescence intensity and the concentration were used to obtain the value of ERF for each model microsphere calibration standard. The procedure is described in detail only for microspheres with allophycocyanin (APC) immobilized on the surface. ERF values were also determined for microsphere calibrators for three other fluorescence channels: fluorescein isothiocyanate (FITC), phycoerythrin (PE), and Pacific Blue(PB). The four model microsphere calibrators provide a one point calibration for the four channels of a flow cytometer. By using software controls and changing the photomultiplier voltages, it is possible to obtain a multipoint calibration for each fluorescence channel using each microsphere calibrator.