Real-time analysis of endosomal lipid transport by live cell scintillation proximity assay.
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.
The Biolaboratories, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
Walter
W
Stockinger
Adam B
AB
Castoreno
Yan
Y
Wang
Joanne C
JC
Pagnon
Axel
A
Nohturfft
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D011992:Endosomes / Q000378:metabolism*; D008027:Light; D050356:Lipid Metabolism; D008247:Lysosomes / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D010587:Phagocytosis; D011374:Progesterone / Q000378:metabolism; D012588:Scintillation Counting / Q000379:methods*; D013997:Time Factors; D014316:Tritium / Q000737:chemistry
D014316:Tritium; D011374:Progesterone; D002784:Cholesterol