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7‐Ketocholesterol favors lipid accumulation and colocalizes with Nile Red positive cytoplasmic structures formed during 7‐ketocholesterol–induced apoptosis: Analysis by flow cytometry, FRET biphoton spectral imaging microscopy, and subcellular fractionation
Author(s) -
Vejux Anne,
Kahn Edmond,
Dumas Dominique,
Bessède Ginette,
Ménétrier Franck,
Athias Anne,
Riedinger JeanMarc,
Frouin Frédérique,
Stoltz JeanFrançois,
OgierDenis Eric,
ToddPokropek Andrew,
Lizard Gérard
Publication year - 2005
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20124
Subject(s) - nile red , flow cytometry , cytoplasm , microbiology and biotechnology , cell fractionation , apoptosis , förster resonance energy transfer , biophysics , cytometry , microscopy , chemistry , biology , biochemistry , fluorescence , physics , optics , membrane
Background Oxidized low‐density lipoproteins play key roles in atherosclerosis. Their toxicity is at least in part due to 7‐ketocholesterol (7KC), which is a potent inducer of apoptosis. In this study on human promonocytic U937 cells, we determined the effects and the interactions of 7KC with cellular lipids during 7KC‐induced apoptosis. Methods Morphologic and functional changes were investigated by microscopic and flow cytometric methods after staining with propidium iodide, 3,3′‐dihexyloxacarbocyanine iodide, and Hoechst 33342. Cellular lipid content was identified by using filipin to quantify free cholesterol and Nile Red (NR), which emit a yellow or orange‐red fluorescence in the presence of neutral and polar lipids, respectively. After staining with NR, interactions of 7KC with cellular lipids were identified by fluorescence resonance energy transfer biphoton spectral imaging confocal microscopy and by subcellular fractionation, gas chromatography, and mass spectrometry. Results During 7KC‐induced apoptosis the fluorescence from filipin and the ratio of measured (orange‐red vs. yellow) fluorescence of NR were enhanced. Spectral analysis of images obtained in biphoton mode and resulting factor images demonstrated the occurrence of fluorescence resonance energy transfer between 7KC and NR and the subsequent colocalization of 7KC and NR. These data were in agreement with biochemical characterization and demonstrated that 7KC and neutral and polar lipids accumulate in NR‐stained cytoplasmic structures. Conclusions During 7KC‐induced apoptosis, 7KC modifies the cellular content of neutral and polar lipids, favors free cholesterol accumulation, and colocalizes with neutral and polar lipids that are inside NR‐stained cytoplasmic structures. © 2005 Wiley‐Liss, Inc.