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Dihydroergosterol is a fluorescent cholesterol analog that it commercially available. It has been reported to be a good measure of the distribution of endogenous cholesterol. Its major drawback is that it is excited around 335nm and its emission peak is around 400nm. Therefore, it requires specialised optical setup for decent imaging.

Filipin works well for fixed cells, but it is important to keep in mind that it requires a minimum concentration of cholesterol to bind. This is around 5 mol% in artificial membranes and means that many intracellular membranes do not show although then contain cholesterol. It is important to saturate the system with filipin, i. e. make sure that increased concentrations or labelling times does not give stronger fluorescence since the fluorescence at low filipin to cholesterol ratios is sensitive to the sphingomyelin concentration.

Regards,

Ingela Parmryd


Ingela Parmryd
Division of Cell Biology
The Wenner-Gren Institute
Stockholm University
106 91 Stockholm
Sweden
Tel: +46 8 16 39 03



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Från: Confocal Microscopy List [mailto:[log in to unmask]] För Shane
Skickat: den 14 april 2008 14:55
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Ämne: Re: Cholesterol dyes

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Filipin III is workable with a 405 nm laser but as pointed out, bleaching can be 
a problem. We've had acceptable results with filipin in fixed cells.

Biotinylated beta-perfringolysin toxin is the only direct method that I know of 
for detecting cholesterol in fixed cells. 

It's not available commercially and despite several requests, I haven't been 
able to get hold of it.

Cholesterol reporters are increasingly important for our work so I may have to 
clone this myself. I'd be happy to make any reagents available.

here's the ref:

Iwamoto M, Morita I, Fukuda M, Murota S, Ando S, Ohno-Iwashita Y. Biochim 
Biophys Acta. 1997 Jul 25;1327(2):222-30. 


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