Quantitative Immunofluorescence to Study Phagosome Maturation.
Cells such as macrophages and neutrophils can internalize a diverse set of particulate matter, illustrated by bacteria and apoptotic bodies through the process of phagocytosis. These particles are sequestered into phagosomes, which then fuse with early and late endosomes, and ultimately with lysosomes to mature into phagolysosomes, through a process known as phagosome maturation. As phagosomes change, they acquire and divest proteins that are associated with the various stages of phagosome maturation. These changes can be assessed at the single-phagosome level by using immunofluorescence methods to study phagosome maturation. Typically, we use indirect immunofluorescence methods that rely on primary antibodies against specific molecular markers that track phagosome maturation. Most commonly, phagosome maturation in macrophages can be determined by staining the cells for Lysosomal-Associated Membrane Protein I (LAMPI) and measuring the fluorescence intensity of LAMPI around each phagosome by microscopy or flow cytometry.
Department of Chemistry and Biology, Ryerson University, 350 Victoria Street, Toronto, ON, Canada, M5B2K3.|The Graduate Program in Molecular Science, Ryerson University, Toronto, ON, Canada, M5B2K3.
Roya M
RM
Dayam
Department of Chemistry and Biology, Ryerson University, 350 Victoria Street, Toronto, ON, Canada, M5B2K3. rbotelho@ryerson.ca.|The Graduate Program in Molecular Science, Ryerson University, Toronto, ON, Canada, M5B2K3. rbotelho@ryerson.ca.
Roberto J
RJ
Botelho
D000818:Animals; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique / Q000379:methods*; D052118:Lysosomal-Associated Membrane Protein 1 / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D010588:Phagosomes / Q000378:metabolism*; D000067996:RAW 264.7 Cells
D052118:Lysosomal-Associated Membrane Protein 1
Antibodies; Immunofluorescence; LAMPI; Lysosomes; Macrophages; Phagosome maturation