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 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.
KeywordsCholesterol endocytosis endosomes lysosomes phagosomes plasma membrane
This work was supported by funding from the Parseghian Medical Research Foundation and the National Institutes of Health (R01 DK59934).
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