An improved flow cytometry assay to monitor phagosome acidification

TY - JOUR

T1 - An improved flow cytometry assay to monitor phagosome acidification

AU - Colas, Chloé

AU - Menezes, Shinelle

AU - Gutiérrez-Martínez, Enric

AU - Péan, Claire B.

AU - Dionne, Marc S.

AU - Guermonprez, Pierre

PY - 2014/10/1

Y1 - 2014/10/1

N2 - 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.

AB - 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.

KW - Flow cytometry

KW - Lysosome

KW - Phagocyte

KW - Phagosome

UR - http://www.scopus.com/inward/record.url?scp=84908306680&partnerID=8YFLogxK

U2 - 10.1016/j.jim.2014.06.008

DO - 10.1016/j.jim.2014.06.008

M3 - Article

AN - SCOPUS:84908306680

SN - 0022-1759

VL - 412

SP - 1

EP - 13

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

ER -