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

Published

June 19, 2011

Author(s)

Hao-Wei Wang, Adolfas Gaigalas

Abstract

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

Download Paper

Local Download

Keywords

multicolor flow cytometer, equivalent reference fluorophores (ERF), fluorescence, fluorescein isothiocyanate (FITC), allophycocyanin (APC), phycoerythrin (PE), pacific blue (PB)

Citation

Wang, H. and Gaigalas, A. (2011), Development of Multicolor Flow Cytometry Standards: Assignment of Equivalent Reference Fluorophores (ERF) Unit, Journal of Research (NIST JRES), National Institute of Standards and Technology, Gaithersburg, MD, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=906632 (Accessed October 13, 2025)