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Quantitative Fluorescence Measurements with Multicolor Flow Cytometry



Lili Wang, Adolfas K. Gaigalas, James Wood


Multicolor flow cytometer assays are routinely used in clinical laboratories for immunophenotyping, monitoring disease and treatment, and determining prognostic factors. However, existing methods for quantitative measurements have not yet produced satisfactory results. This chapter details a procedure for quantifying surface and intracellular protein biomarkers by calibrating the output of a multicolor flow cytometer in units of antibodies bound per cell (ABC). The procedure includes the following critical steps, (a) quality control (QC) and performance characterization of the multicolor flow cytometer, (b) fluorescence calibration using hard dyed microspheres assigned with fluorescence intensity values in equivalent number of reference fluorophores (ERF), (c) compensation for correction of fluorescence spillover, and (d) application of a biological reference standard for translating the ERF scale to the ABC scale. The chapter also points out current efforts for implementing quantification of biomarkers in a manner which is independent of instrument platforms and reagent differences.
Flow Cytometry Protocols
Publisher Info
Springer Science + Business Media, LLC, New York, NY


Multicolor flow cytometry, Fluorescence calibration, Equivalent number of reference fluorophores (ERF), CD4+ lymphocytes, Antibodies bound per cell, Instrument quality control, Instrument sensitivity, Pulsed light-emitting diode (LED) light source, Compensation.


Wang, L. , Gaigalas, A. and Wood, J. (2017), Quantitative Fluorescence Measurements with Multicolor Flow Cytometry, Flow Cytometry Protocols, Springer Science + Business Media, LLC, New York, NY (Accessed May 21, 2024)


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Created November 20, 2017, Updated January 25, 2018