Open AccessDynamic Quantitative Assays of Phagosomal Function
Author(s) -
Podinovskaia Maria,
VanderVen Brian C.,
Yates Robin M.,
Glennie Sarah,
Fullerton Duncan,
Mwandumba Henry C.,
Russell David G.
Publication year - 2013
Publication title -
current protocols in immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.618
H-Index - 43
eISSN - 1934-368X
pISSN - 1934-3671
DOI - 10.1002/0471142735.im1434s102
Subject(s) - phagosome , flow cytometry , lysosome , chemistry , förster resonance energy transfer , effector , macrophage , microbiology and biotechnology , superoxide , confocal microscopy , biophysics , respiratory burst , intracellular , fluorescence , biochemistry , biology , in vitro , physics , quantum mechanics , enzyme
Much of the activity of the macrophage as an effector cell is performed within its phagocytic compartment. This ranges from the degradation of tissue debris as part of its homeostatic function to the generation of the superoxide burst as part of its microbicidal response to infection. We have developed a range of real‐time readouts of phagosomal function that enable these activities to be rigorously quantified. This unit contains descriptions of several of these assays assessed by different methods of quantitation, including a fluorescence resonance emission transfer (FRET) assay for phagosome/lysosome fusion measured by spectrofluorometry, a fluorogenic assay for the superoxide burst measured by flow cytometry, and a fluorogenic assay for bulk proteolysis measured by confocal microscopy. These assays illustrate both the range of parameters that can be quantified and the flexibility of instrumentation that can be exploited for their quantitation. Curr. Protoc. Immunol . 102:14.34.1‐14.34.14. © 2013 by John Wiley & Sons, Inc.