A procedure is described for assigning values of the number of equivalent reference fluorophores (ERF) to microspheres labeled with a fluorophore designed to produce fluorescence in a given channel of a multicolor flow cytometer. There is a different microsphere for each of the fluorescence channels. The assignment of the ERF value utilized a fluorimeter which had been calibrated by a series of solutions of the reference fluorophore. The fluorimeter was used to obtain microsphere fluorescence intensity, and a multicolor flow cytometer was used to obtain microsphere concentration. The fluorescence intensity and the concentration were used to obtain a value of ERF. The procedure is described in detail for microspheres with APC immobilized on the surface. ERF values were also determined for microspheres for three other fluorescence channels: FITC, PE, and PB. The four microspheres individually provide a one point calibration for four channels of a flow cytometer. By changing the photomultiplier voltage, it is possible to obtain a multipoint calibration using only one microsphere population.
Citation: Journal of Research (NIST JRES) -
NIST Pub Series: Journal of Research (NIST JRES)
Pub Type: NIST Pubs
multicolor flow cytometer, equivalent reference fluorophores (ERF), fluorescence, fluorescein isothiocyanate (FITC), allophycocyanin (APC), phycoerythrin (PE), pacific blue (PB)