Abstract
The biochemical processes within the phagosomes of macrophages and dendritic cells are essential to immunity and homeostasis. Measurable properties of the phagosomal lumen include: assessment of various hydrolytic activities, reduction and oxidation events, pH, ion concentrations, and electrochemical gradients. These often-interdependent phagosomal features are commonly evaluated individually, hindering the analysis of the biochemical relationship between these factors within the same phagosome. In addition, the ability of phagosomes within the same cell to behave autonomously is becoming more evident, thus highlighting the need for a technique capable of multiplex analyses of phagosomal lumenal chemistries at the single phagosome level. In this chapter, we outline an approach that is capable of simultaneously measuring multiple phagosomal parameters of individual phagosomes by utilizing specifically designed experimental particles with multiple reporter fluors, in combination with real-time fluorometric measurement via automated microscopy. Subsequent analysis using high-content image analysis software enables each phagosomal parameter to be evaluated in a quantitative or semi-quantitative manner. This approach facilitates investigation of the complex relationship between phagosomal properties in a population of macrophages in real time, at the level of individual phagosomes.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Vieira OV, Botelho RJ, Grinstein S (2002) Phagosome maturation: aging gracefully. Biochem J 366:689–704
Kinchen JM, Ravichandran KS (2008) Phagosome maturation: going through the acid test. Nat Rev Mol Cell Biol 9:781–795
Desjardins M, Huber LA, Parton RG et al (1994) Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus. J Cell Biol 124:677–688
Schlam D, Bohdanowicz M, Chatgilialoglu A et al (2013) Diacylglycerol kinases terminate diacylglycerol signaling during the respiratory burst leading to heterogeneous phagosomal NADPH oxidase activation. J Biol Chem 288:23090–23104
VanderVen BC, Yates RM, Russell DG (2009) Intraphagosomal measurement of the magnitude and duration of the oxidative burst. Traffic 10:372–378
Yates RM, Hermetter A, Russell DG (2009) Recording phagosome maturation through the real-time, spectrofluorometric measurement of hydrolytic activities. Methods Mol Biol 531:157–171
Yates RM, Russell DG (2008) Real-time spectrofluorometric assays for the lumenal environment of the maturing phagosome. Methods Mol Biol 445:311–325
Rybicka JM, Balce DR, Khan MF et al (2010) NADPH oxidase activity controls phagosomal proteolysis in macrophages through modulation of the lumenal redox environment of phagosomes. Proc Natl Acad Sci U S A 107:10496–10501
Rybicka JM, Balce DR, Chaudhuri S et al (2012) Phagosomal proteolysis in dendritic cells is modulated by NADPH oxidase in a pH-independent manner. EMBO J 31:932–944
Balce DR, Yates RM (2013) Redox-sensitive probes for the measurement of redox chemistries within phagosomes of macrophages and dendritic cells. Redox Biol 1:467–474
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Cheung, S., Greene, C., Yates, R.M. (2017). Simultaneous Analysis of Multiple Lumenal Parameters of Individual Phagosomes Using High-Content Imaging. In: Botelho, R. (eds) Phagocytosis and Phagosomes. Methods in Molecular Biology, vol 1519. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6581-6_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6581-6_15
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6579-3
Online ISBN: 978-1-4939-6581-6
eBook Packages: Springer Protocols