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Updated: May 19, 2026

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization
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High-throughput phagocytosis assay utilizing a pH-sensitive fluorescent dye.

Anton Beletskii1, Michael Cooper, Priya Sriraman

  • 1GE Global Research Center, Niskayuna, NY 12309, USA anton.beletskii@ge.com

Biotechniques
|December 31, 2005
PubMed
Summary

A novel assay using the pH-sensitive dye CypHer5E enables high-throughput measurement of phagocytosis. This method accurately quantifies particle uptake by cells, distinguishing it from mere adherence.

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Area of Science:

  • Cell Biology
  • Immunology
  • Biochemistry

Background:

  • Phagocytosis is a critical cellular process for immune response and cellular homeostasis.
  • Existing phagocytosis assays often suffer from high background noise and difficulty distinguishing uptake from adherence.
  • There is a need for robust, high-throughput assays to screen compounds affecting phagocytosis.

Purpose of the Study:

  • To develop and validate a novel, high-throughput phagocytosis assay using a pH-sensitive cyanine dye, CypHer5E.
  • To demonstrate the assay's reliability across various cell types and its suitability for screening applications.
  • To compare the advantages of the CypHer5E assay over traditional methods.

Main Methods:

  • Utilized CypHer5E, a pH-sensitive cyanine dye that fluoresces in acidic environments, for labeling particles.

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  • Applied the assay to various cell types, including primary human monocytes, dendritic cells, endothelial cells, and cell lines (THP-1, WBC264-9C).
  • Performed assays in 96- and 384-well formats and determined the IC50 for cytochalasin D inhibition of particle uptake.

    • Main Results:

    • The CypHer5E assay provides reduced background fluorescence from non-internalized particles.
    • CypHer5E-labeled particles exhibit resistance to fluorescence quenching in the phagosome.
    • The assay demonstrated reliable performance across diverse primary cells and cell lines, distinguishing true phagocytosis from adherence.

    Conclusions:

    • The CypHer5E-based phagocytosis assay is a robust and sensitive method for high-throughput screening.
    • This assay offers significant advantages over existing protocols, including improved accuracy and suitability for microscopy.
    • The developed assay facilitates the study of cellular processes and compound effects on phagocytosis.