Quantitative Live-Cell Fluorescence Microscopy During Phagocytosis.
Phagocytosis is a receptor-mediated process whereby professional phagocytes internalize invading pathogens and apoptotic bodies into an intracellular vacuole or phagosome, leading to their degradation. During the formation and maturation of the phagosome, several lipids undergo changes and effector proteins are recruited on the nascent phagosome in a concerted manner. These highly localized, dynamic, and transient processes can only be studied by methods capable of high spatial and temporal resolution. The use of genetically encoded chimeric constructs coupled with fluorescence confocal microscopy enables the continuous, noninvasive analysis of the distribution and metabolism of lipids and effector proteins during phagocytosis. Here, we describe a method where the mouse macrophage cell line, RAW 264.7, and primary macrophages are transiently transfected with fluorescent chimeric probes to analyze and quantify phagocytosis of immunoglobulin-opsonized particles, using confocal microscopy.
Department of Biochemistry, University of Toronto, Toronto, ON, Canada, M5S 1A8.|Program in Cell Biology, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children, 686 Bay Street, 19-9800, Toronto, ON, Canada, M5G 0A4.|Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael's Hospital, 290 Victoria Street, Toronto, ON, Canada, M5C 1N8.
Stella M
SM
Lu
Program in Cell Biology, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children, 686 Bay Street, 19-9800, Toronto, ON, Canada, M5G 0A4.|Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael's Hospital, 290 Victoria Street, Toronto, ON, Canada, M5C 1N8.
Sergio
S
Grinstein
Department of Biochemistry, University of Toronto, Toronto, ON, Canada, M5S 1A8. fairng@smh.ca.|Program in Cell Biology, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children, 686 Bay Street, 19-9800, Toronto, ON, Canada, M5G 0A4. fairng@smh.ca.|Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael's Hospital, 290 Victoria Street, Toronto, ON, Canada, M5C 1N8. fairng@smh.ca.
Gregory D
GD
Fairn
D000818:Animals; D018929:Cell Culture Techniques; D002469:Cell Separation; D002470:Cell Survival; D018274:Electroporation; D007091:Image Processing, Computer-Assisted; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D010587:Phagocytosis; D000067996:RAW 264.7 Cells; D014162:Transfection
Confocal microscopy; Fluorescent probes; Macrophage; Phagocytosis