Studying phagocytosis by live-cell scintillation proximity assay.
Phagocytosis of microorganisms, senescent cells, apoptotic bodies, and effete tissue material is an important process in host defense and tissue homeostasis. A method is described to measure, in living macrophages, the kinetics of particle engulfment and lysosome/phagosome targeting. Plasma membranes or lysosomes are labeled with tritiated lipids, followed by exposure of cells to scintillant microbeads. Because of the short range of tritium beta-particles, geometric factors, and the confinement of lipids to membranes, scintillation can only be elicited by tracer molecules in membranes immediately vicinal to the scintillant. When the plasma membrane.is labeled with [(3)H]cholesterol, a signal is produced on bead-cell contact and engulfment and then reaches steady state within 45 min. When lysosomes are labeled with nonhydrolyzable [(3)H]cholesterol oleyl ether, scintillation requires intracellular lysosome/phagosome attachment or fusion, and steady state is attained only after several hours. The live-cell scintillation proximity approach is useful for examining the effects of pharmacological and genetic manipulations on particle uptake and on lysosome/phagosome targeting.
Stockinger
Walter
W
Max F. Perutz Laboratories, Vienna, Austria.
Nohturfft
Axel
A
D000818:Animals; D002460:Cell Line; D002784:Cholesterol / Q000031:analogs & derivatives / Q000378:metabolism*; D003627:Data Interpretation, Statistical; D007700:Kinetics; D008081:Liposomes; D008247:Lysosomes / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D008561:Membrane Fusion; D051379:Mice; D010587:Phagocytosis; D010588:Phagosomes / Q000378:metabolism; D012588:Scintillation Counting / Q000706:statistics & numerical data; D014316:Tritium
D008081:Liposomes; D014316:Tritium; C023511:cholesteryl oleyl ether; D002784:Cholesterol