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Journal of Lipid Research, Vol. 45, 2151-2158, November 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

Methods

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 Nohturfft¹

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

¹ To whom correspondence should be addressed. e-mail: axno{at}mcb.harvard.edu

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 ß particles by the scintillant produces light signals that can be measured in standard scintillation counters. Because of the short range of tritium ß particles and for geometric reasons, scintillant microspheres detect only that fraction of tritiated cholesterol localized inside phagolysosomes or within a distance of 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.

Abbreviations: AcLDL, acetylated LDL; CO, cholesteryl oleate; SPA, scintillation proximity assay; YSi, yttrium silicate

Supplementary key words cholesterol transport • endosomes • lysosomes • macrophages • phagocytosis

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