Fluorescence-intensity multiplexing: simultaneous seven-marker, two-color immunophenotyping using flow cytometry

Cytometry. Part a : the Journal of the International Society for Analytical Cytology
Jolene A BradfordJoseph M Beechem

Abstract

Conventional immuno-based multiparameter flow cytometric analysis has been limited by the requirement of a dedicated detection channel for each antibody-fluorophore set. To address the need to resolve multiple biological targets simultaneously, flow cytometers with as many as 10-15 detection channels have been developed. In this study, a new Zenon immunolabeling technology is developed that allows for multiple antigen detection per detection channel using a single fluorophore, through a unique method of fluorescence-intensity multiplexing. By varying the Zenon labeling reagent-to-antibody molar ratio, the fluorescence intensity of the antibody-labeled cellular targets can be used as a unique identifier. Although demonstrated in the present study with lymphocyte immunophenotyping, this approach is broadly applicable for any immuno-based multiplexed flow cytomety assay. Lymphocyte immunophenotyping of 38 clinical blood specimens using CD3, CD4, CD8, CD16, CD56, CD19, and CD20 antibodies was performed using conventional flow cytometric analysis and fluorescence-intensity multiplexing analysis. Conventional analysis measures a single antibody-fluorophore per photomultiplier tube (PMT). Fluorescence-intensity multiplex analysis simu...Continue Reading

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Citations

Aug 25, 2006·Nature Methods·Michael G KattahPaul J Utz
Jul 3, 2010·Nature Protocols·Christina BasfordColin McGuckin
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Apr 5, 2014·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Cameron A SmurthwaiteRoland Wolkowicz
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Jul 31, 2013·Journal of Biomolecular Screening·Paolo Cappella, Fabio Gasparri
Mar 26, 2015·Journal of Biomolecular Screening·Bruce S Edwards, Larry A Sklar

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