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PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1984-4-13
pubmed:abstractText
The polyene antibiotic filipin, which forms specific complexes with 3 beta-hydroxysterols, displays spectral properties compatible with its use in flow microfluorometry (FMF). The purpose of this study was to test the suitability of filipin as an FMF probe for unesterified cellular cholesterol. The following experimental conditions appeared optimal for cells with an average unesterified cholesterol content of less than 3 nmol per 10(6) cells: 2 X 10(6) fixed cells (1-4% p-formaldehyde, 30 min, 21 degrees C) stained for 2-4 h with 100 micrograms/ml filipin and excited at 350.7/356.7 nm. Fluorescence emission (Em) was measured above 510 nm. Less suitable conditions involved excitation at 488 nm or using cells which had not been fixed. Fixation preserved the live-dead cell discrimination provided by forward light scatter measurements, so that dead cells could be excluded from the FMF analysis of cellular cholesterol. Under the above conditions FMF analysis of a variety of murine cell types showed that in all cases the fluorescence intensity of filipin-stained cells was clearly increased above autofluorescence levels of the unstained control cells. The increase in fluorescence signal in different filipin stained cell types correlated (P less than or equal to .001) with the cellular content of unesterified cholesterol determined by an independent enzymatic assay. The sensitivity of the FMF assay was in the femtomole (10(-15) ) range. Mixing experiments with cells of different cholesterol levels showed that the technique distinguishes cell populations with distinctive levels of unesterified cholesterol. We therefore concluded that filipin is a useful FMF probe for determining relative levels of unesterified cholesterol in cells.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0196-4763
pubmed:author
pubmed:issnType
Print
pubmed:volume
5
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
42-54
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1984
pubmed:articleTitle
Filipin as a flow microfluorometry probe for cellular cholesterol.
pubmed:publicationType
Journal Article, Comparative Study