Skip to main content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Quantitative Fluorescence Measurements with Multicolor Flow Cytometry

Published

Author(s)

Lili Wang, Adolfas K. Gaigalas, James Wood

Abstract

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.
Citation
Flow Cytometry Protocols
Publisher Info
Springer Science + Business Media, LLC, New York, NY

Keywords

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.
Created November 20, 2017, Updated January 25, 2018