R. Pagano
1989
Citations
4
Influential Citations
80
Citations
Quality indicators
Journal
Methods in cell biology
Abstract
Publisher Summary This chapter discusses the synthesis and purification of C 6 -NBD ceramide and its application in the study of the Golgi apparatus of animal cells. When cells are treated with c,-NBD-ceramide at low temperature, washed, and warmed to 37°C, the Golgi apparatus and later the plasma membrane becomes intensely fluorescent. During the redistribution of intracellular fluorescence, the C 6 -NBD-ceramide is metabolized to fluorescent sphingomyelin and glucosylceramide. Consistent with the increasing fluorescence at the plasma membrane over time, increasing amounts of each fluorescent metabolite can be removed from the cell surface by back-exchange to nonfluorescent acceptor liposomes. Commercial sources of D - erythro -sphingosine can be contaminated with significant amounts of L -threo-sphingosine, resulting in two isomers of C 6 -NBD-ceramide being produced during synthesis. Resonance energy transfer (RET) between C,-NBD-labeled lipids and Rhodamine B-labeled phosphatidylethanol-amine (N-Rh-PE) can be used to monitor the rate of transfer of C,-NBD-labeled lipids between two populations of lipid vesicles. It is found that RET can also be used to test the ability of a C,-NBD-lipid to undergo transbilayer movement in unilamellar vesicles.