Abstract
A scintillation proximity assay has been developed to study the endosomal trafficking of radiolabeled cholesterol in living cells. Mouse macrophages were cultured in the presence of tritiated cholesterol and scintillant microspheres. Microspheres were taken up by phagocytosis and stored in phagolysosomes. Absorption of tritium beta particles by the scintillant produces light signals that can be measured in standard scintillation counters. Because of the short range of tritium beta particles and for geometric reasons, scintillant microspheres detect only that fraction of tritiated cholesterol localized inside phagolysosomes or within a distance of approximately 600 nm. By incubating cultures in a temperature-controlled microplate reader, the kinetics of phagocytosis and cholesterol transport could be analyzed in near-real time. Scintillation signals were significantly increased in response to inhibitors of lysosomal cholesterol export. This method should prove a useful new tool for the study of endosomal trafficking of lipids and other molecules.
Highlights
A scintillation proximity assay has been developed to study the endosomal trafficking of radiolabeled cholesterol in living cells
scintillation proximity assay (SPA) is based in part on the fact that low-energy  particles have a high propensity to interact with matter and propagate only short distances
If decaying atoms are localized in sufficient proximity to a scintillating microsphere, electron absorption by the scintillant may lead to photon emission, which can be detected by a scintillation counter
Summary
A scintillation proximity assay has been developed to study the endosomal trafficking of radiolabeled cholesterol in living cells. Scintillation signals were significantly increased in response to inhibitors of lysosomal cholesterol export This method should prove a useful new tool for the study of endosomal trafficking of lipids and other molecules.—Stockinger, W., A. Real-time analysis of endosomal lipid transport by live cell scintillation proximity assay. Cholesterol is synthesized from acetate or enters cells as a component of serum-derived lipoproteins by endocytosis Lipoprotein particles such as LDL carry cholesterol predominantly as cholesteryl acyl ester that is hydrolyzed by the endosomal/lysosomal enzyme acidic cholesteryl ester hydrolase, liberating cholesterol and fatty acid [1]. Two-thirds of endosomally derived cholesterol are transported to the plasma membrane and the other one-third is thought to be moved along an independent pathway to the endoplasmic reticulum (ER) [2]. Cholesterol levels at the cell surface can be measured after cell fixation based on the sensitivity of plasma mem-
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