Flow cytometry and factor analysis evaluation of confocal image sequences of morphologic and functional changes occurring at the mitochondrial level during 7-ketocholesterol-induced cell death.
OBJECTIVE
To analyze functional and morphologic alterations that occur at the mitochondrial level by flow cytometry and laser scanning confocal microscopy (CLSM) combined with factor analysis of biomedical image sequences (FAMIS).


METHODS
Under treatment of U937 cells with 7-ketocholesterol, functional alterations that occur at the mitochondrial level (especially loss of transmembrane mitochondrial potential [delta psi m]) were assessed with 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) and mitotracker red (CMXRos), whereas morphologic changes were analyzed with nonyl acridine orange (NAO). By flow cytometry, these different dyes were excited at 488 nm, whereas on CLSM, excitation of NAO and CMXRos was performed by lines of an argon laser. By CLSM, spectral sequences were performed to characterize NAO and CMXRos. FAMIS was used to transform the image sequences in factor images.


RESULTS
By flow cytometry, rapid loss of delta psi m induced by 7-ketocholesterol was detected with both DiOC6(3) and CMXRos, which gave similar results. Morphologic alterations of mitochondria were revealed with NAO. The factor images obtained from confocal image sequences confirmed these results.


CONCLUSIONS
The simultaneous use of NAO, CMXRos and FAMIS constitutes a new method to detect morphologic and functional alterations occurring at the mitochondrial level during cell death.
Kahn
Edmond
E
National Institute of Health and Medical Research U494, Centre Hospitalier Universitaire Pitié Salpêtrière, Paris, France. kahn@imed.jussieu.fr
Lizard
Gérard
G
Monier
Serge
S
Bessède
Ginette
G
Frouin
Frédérique
F
Gambert
Philippe
P
Todd-Pokropek
Andrew
A
D016923:Cell Death; D002478:Cells, Cultured; D005163:Factor Analysis, Statistical; D005434:Flow Cytometry; D006801:Humans; D007653:Ketocholesterols / Q000633:toxicity*; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000502:physiology* / Q000648:ultrastructure*; D020298:U937 Cells
D007653:Ketocholesterols; C003001:7-ketocholesterol