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Related Experiment Video

Updated: Mar 28, 2026

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy
14:39

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Published on: December 7, 2015

14.5K

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy.

Paula Nunes1, Daniele Guido2, Nicolas Demaurex2

  • 1Department of Cellular Physiology and Metabolism, University of Geneva; Paula.Nunes@unige.ch.

Journal of Visualized Experiments : Jove
|December 29, 2015
PubMed
Summary
This summary is machine-generated.

This study introduces a novel ratiometric imaging method to measure phagosomal pH in neutrophils. This technique accurately quantics phagosome acidification, crucial for immune cell function and pathogen defense.

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Area of Science:

  • Immunology
  • Cell Biology
  • Biochemistry

Background:

  • Phagocytosis is a key innate immune process for clearing pathogens and cellular debris.
  • Phagosomal pH is critical for phagosome maturation, degradation, and immune signaling.
  • Intracellular pathogens often manipulate phagosomal pH to evade immune responses.

Purpose of the Study:

  • To develop and validate a ratiometric imaging method for precise measurement of phagosomal pH in neutrophils.
  • To provide a generalized protocol for functional imaging of phagosomes in various cell types and with different targets.

Main Methods:

  • Utilized fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets in neutrophils.
  • Employed a single-dye ratiometric fluorescence imaging technique based on FITC's pH-dependent fluorescence properties.
  • Developed an in situ dye calibration protocol for accurate conversion of fluorescence ratios to pH values.

Main Results:

  • The ratiometric method allows for accurate quantification of phagosomal pH by measuring a pH-dependent fluorescence ratio.
  • This single-dye approach minimizes artifacts from photobleaching, focus changes, and laser variations.
  • The protocol is adaptable for other phagocytic cells and targets, including microorganisms.

Conclusions:

  • The developed ratiometric method offers a robust and versatile tool for studying phagosomal pH dynamics.
  • Accurate phagosomal pH measurement is vital for understanding immune cell function and host-pathogen interactions.
  • This generalized protocol facilitates further research into phagosome biology and the development of novel therapeutic strategies.