An improved flow cytometry assay to monitor phagosome acidification

Phago-lysosome formation is important for cell-autonomous immunity to intracellular pathogens, antigen presentation and metabolism. A hallmark feature of phago-lysosomal compartments is that they undergo progressive luminal acidification controlled by the activation of vacuolar V-ATPase. Acidificati...

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Bibliographic Details
Published in:Journal of immunological methods 2014-10, Vol.412, p.1-13
Main Authors: Colas, Chloé, Menezes, Shinelle, Gutiérrez-Martínez, Enric, Péan, Claire B., Dionne, Marc S., Guermonprez, Pierre
Format: Article
Language:English
Subjects:
Animals
Cell Line
Cell Separation - methods
Flow cytometry
Flow Cytometry - methods
Fluorescence
Hydrogen-Ion Concentration
Lysosome
Lysosomes - enzymology
Macrophages - immunology
Mice
Mice, Inbred C57BL
Microspheres
Phagocyte
Phagocytosis
Phagosome
Phagosomes - enzymology
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Summary:Phago-lysosome formation is important for cell-autonomous immunity to intracellular pathogens, antigen presentation and metabolism. A hallmark feature of phago-lysosomal compartments is that they undergo progressive luminal acidification controlled by the activation of vacuolar V-ATPase. Acidification is required for many enzymatic processes taking place in phago-lysosomes, like proteolysis, and supports the microbicidal activity of macrophages. Here we present a new quantitative methodology to assess phagosome acidification by flow cytometry based on the use of bi-fluorescent particles. This method relies on the use of UV polystyrene beads labelled with the acid sensor pHrodo-succinimidyl ester (pHrodoTM SE red) and enables us to dissociate particle association with phagocytes from their engulfment in acidified compartments. This methodology is well suited to monitor the acidification of phagosomes formed in vivo after fluorescent bead administration. •Bi-Fluorescent latex beads coated with pHrodo pH monitor phago-lysosome formation by FACS•The sensor detects independently bead association to cells and phagosome acidification.•The sensor is compatible with multi-parameter flow cytometry and can be used in vivo.
ISSN:0022-1759
1872-7905
DOI:10.1016/j.jim.2014.06.008