title: An improved flow cytometry assay to monitor phagosome acidification.
abstract: 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 (pHrodo(TM) 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.
authors: Phagocyte Immunobiology Laboratory, Centre for Molecular and Cellular Biology of Inflammation, Peter Gorer Department of Immunobiology, Division of Immunology, Infection & Inflammatory Diseases, King's College London, United Kingdom.
authors: Chloé
authors: C
authors: Colas
authors: Phagocyte Immunobiology Laboratory, Centre for Molecular and Cellular Biology of Inflammation, Peter Gorer Department of Immunobiology, Division of Immunology, Infection & Inflammatory Diseases, King's College London, United Kingdom.
authors: Shinelle
authors: S
authors: Menezes
authors: Phagocyte Immunobiology Laboratory, Centre for Molecular and Cellular Biology of Inflammation, Peter Gorer Department of Immunobiology, Division of Immunology, Infection & Inflammatory Diseases, King's College London, United Kingdom.
authors: Enric
authors: E
authors: Gutiérrez-Martínez
authors: Immunometabolism Laboratory, Centre for Molecular and Cellular Biology of Inflammation, Peter Gorer Department of Immunobiology, Division of Immunology, Infection & Inflammatory Diseases, King's College London, United Kingdom.
authors: Claire B
authors: CB
authors: Péan
authors: Immunometabolism Laboratory, Centre for Molecular and Cellular Biology of Inflammation, Peter Gorer Department of Immunobiology, Division of Immunology, Infection & Inflammatory Diseases, King's College London, United Kingdom.
authors: Marc S
authors: MS
authors: Dionne
authors: Phagocyte Immunobiology Laboratory, Centre for Molecular and Cellular Biology of Inflammation, Peter Gorer Department of Immunobiology, Division of Immunology, Infection & Inflammatory Diseases, King's College London, United Kingdom. Electronic address: pierre.guermonprez@kcl.ac.uk.
authors: Pierre
authors: P
authors: Guermonprez
mesh_terms: D000818:Animals; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000201:enzymology*; D008264:Macrophages / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D008863:Microspheres; D010587:Phagocytosis; D010588:Phagosomes / Q000201:enzymology*
keywords: Flow cytometry; Lysosome; Phagocyte; Phagosome