Analysis of the fluorescence of monodansylcadaverine-positive cytoplasmic structures during 7-ketocholesterol-induced cell death.
OBJECTIVE
To analyze multilamellar cytoplasmic structures by confocal laser scanning microscopy (CLSM) combined with factor analysis of biomedical image sequences (FAMIS).


METHODS
After treatment of U937 cells with 7-ketocholesterol (7-keto), cytoplasmic alterations were assessed with monodansylcadaverine (MDC). By ultraviolet excitation of a confocal laser scanning microscope (UV-CLSM), spectral sequences were performed to characterize 7-keto and MDC distribution inside cells. FAMIS was used to transform the image sequences in factor curves and images.


RESULTS
By UV-CLSM, 7-keto fluorescence was detected together with MDC, which revealed morphologic cytoplasmic changes in cells. The factor images obtained from confocal image sequences emphasized the view of these results. These data are in agreement with biochemical characterizations of MDC-positive structures.


CONCLUSIONS
The combined use of confocal microscopy and FAMIS allowed us to detect MDC-positive cytoplasmic structures in 7-keto-treated cells and to colocalize MDC and 7-keto distribution. This new method confirms the usefulness of MDC as a marker of oxysterol-induced cell death.
Kahn
Edmond
E
Institut National de la Santé et de la Recherche Médicale U494, Centre Hospitalier Universitaire Pitié-Salpêtrière, 75634 Paris, France. kahn@imed.jussieu.fr
Vejux
Anne
A
Lizard
Gérard
G
Bessède
Ginette
G
Frouin
Frédérique
F
Prunet
Céline
C
Bernengo
Jean-Claude
JC
Brau
Frédéric
F
Todd-Pokropek
Andrew
A
Gambert
Philippe
P
D001562:Benzimidazoles / Q000737:chemistry; D002103:Cadaverine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000494:pharmacology; D002232:Carbocyanines / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D002467:Cell Nucleus / Q000737:chemistry / Q000187:drug effects; D002499:Centrifugation, Density Gradient; D022083:Cytoplasmic Structures / Q000737:chemistry* / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007653:Ketocholesterols / Q000032:analysis / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000737:chemistry / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D020298:U937 Cells
D001562:Benzimidazoles; D002232:Carbocyanines; D004791:Enzyme Inhibitors; D007653:Ketocholesterols; C007392:3,3'-dihexyl-2,2'-oxacarbocyanine; C008542:monodansylcadaverine; D002103:Cadaverine; C003001:7-ketocholesterol; C017807:bisbenzimide ethoxide trihydrochloride