Measuring pH, ROS production, maturation, and degradation in dendritic cell phagosomes using cytofluorometry-based assays.
Phagosomes are complex organelles that form after ingestion by phagocytic cells of pathogens, dying cells, or cell debris. Highly dynamic interactions of phagosomes first with endosomes and then with lysosomes lead to the maturation of phagosomes into phagolysosomes. Contrary to other phagocytes, which degrade ingested particles to amino acids, dendritic cells only partially degrade ingested proteins, preserving short peptides for the onset of adaptive immune responses. We have modified a series of latex bead-based techniques, previously reported, in order to analyze phagosome maturation using flow cytometry. The analysis of the phagosomal pH, degradation, or oxidation relies on techniques based on the fate of specific probes bound to particles to be phagocytosed. These techniques are very sensitive and quantitative.
Savina
Ariel
A
Institut Curie, INSERM U653, Immunité et Cancer, Paris, France.
Vargas
Pablo
P
Guermonprez
Pierre
P
Lennon
Ana-Maria
AM
Amigorena
Sebastian
S
D000818:Animals; D003713:Dendritic Cells / Q000166:cytology* / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D010588:Phagosomes / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*
D017382:Reactive Oxygen Species