J. Lipid Res.
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A more recent version of this article appeared on November 1, 2004

Papers In Press, published online ahead of print August 16, 2004
J. Lipid Res., doi:10.1194/jlr.D400011-JLR200
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Submitted on June 21, 2004
Revised on August 16, 2004
Accepted on July 28, 2004

Real time analysis of endosomal lipid transport by live cell scintillation proximity assay

Walter Stockinger, Adam B. Castoreno, Yan Wang, Joanne C. Pagnon, and Axel Nohturfft

Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138

Corresponding Author: axno{at}mcb.harvard.edu

A scintillation proximity assay has been developed to study 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. Due to 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 about 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. The method should prove a useful new tool to study endosomal trafficking of lipids and other molecules.


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