Measurement of bacterial capture and phagosome maturation of Kupffer cells by intravital microscopy

Methods : a Companion to Methods in Enzymology
Bas G J Surewaard, Paul Kubes

Abstract

It is central to the field of bacterial pathogenesis to define how bacteria are killed by phagocytic cells. During phagocytosis, the microbe is localized to the phagolysosome where crucial defense mechanisms such as acidification and production of reactive oxygen species (ROS) are initiated. This process has extensively been studied in vitro, however many resident tissue phagocytes will phenotypically change upon isolation from their natural environment. Therefore, interrogation of phagocytosis and phagosomal function of cells in the context of their natural tissue environment enhances our understanding of the biological process in vivo. This article outlines a real-time intravital microscopy protocol that utilizes fluorescent dyes to study the process of phagocytosis, which reveals acidification and oxidation of individual bacteria inside host cells of living animals. The novelty of this technique exists in use of bacteria that are covalently labelled with the fluorescent dyes Oxyburst and pHrodo, which respectively report on oxidation or acidification. Intravital microscopy is applied to visualize the uptake and subsequent oxidation or acidification of reporter bacteria in the organ of interest. Fluorescently labelled antibod...Continue Reading

Citations

Feb 19, 2019·Journal of Leukocyte Biology·Brittney N V ScottAjitha Thanabalasuriar
Jan 25, 2020·The Journal of Experimental Medicine·Carsten DeppermannPaul Kubes
Dec 6, 2017·Cell and Tissue Research·Megan L Lewis, Bas G J Surewaard
Jun 7, 2019·Hepatology Communications·Adrien Guillot, Frank Tacke
Nov 17, 2020·Frontiers in Cellular and Infection Microbiology·Ann-Britt Schäfer, Michaela Wenzel
Feb 6, 2021·Frontiers in Immunology·Grace R PidwillSimon J Foster
Aug 8, 2020·Hepatology : Official Journal of the American Association for the Study of Liver Diseases·Carsten DeppermannMamatha Bhat
Apr 18, 2021·Cellular & Molecular Immunology·Ola AhmedCliona O'Farrelly
Mar 9, 2021·American Journal of Physiology. Renal Physiology·Kenneth W DunnPierre C Dagher

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