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 β-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 [ 3H]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 [3H]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. © 2008 Humana Press.
CITATION STYLE
Stockinger, W., & Nohturfft, A. (2008). Studying phagocytosis by live-cell scintillation proximity assay. Methods in Molecular Biology, 440, 147–155. https://doi.org/10.1007/978-1-59745-178-9_11
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