The application of fluorescent probes for the analysis of lipid dynamics during phagocytosis.
Phagocytosis is the process whereby specialized leukocytes ingest large particles. This is an extremely dynamic and localized process that requires the recruitment to the sites of ingestion of numerous effector proteins, together with extensive lipid remodelling. To investigate such a dynamic series of events in living cells, non-invasive methods are required. The use of fluorescent probes in conjunction with spectroscopic analysis is optimally suited for this purpose. Here we describe a method to express in RAW264.7 murine macrophages genetically encoded probes that allow for the spatio-temporal analysis of lipid distribution and metabolism during phagocytosis of immunoglobulin-opsonized beads. The fluorescence of the probes is best analysed by laser scanning or spinning disc confocal microscopy. While the focus of this chapter is on phagocytic events, this general method can be employed for the analysis of lipid distribution and dynamics during a variety of biological processes in the cell type of the investigator's choice.
Flannagan
Ronald S
RS
Program in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada.
Grinstein
Sergio
S
D000818:Animals; D002460:Cell Line; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D050356:Lipid Metabolism; D051379:Mice; D018613:Microscopy, Confocal; D057054:Molecular Imaging; D010587:Phagocytosis; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014162:Transfection
D005456:Fluorescent Dyes