Confocal analysis of phosphatidylserine externalization with the use of biotinylated annexin V revealed with streptavidin-FITC, -europium, -phycoerythrin or -Texas Red in oxysterol-treated apoptotic cells.
OBJECTIVE
To analyze externalization of phosphatidylserine via annexin V on apoptotic cells by laser scanning confocal microscopy and factor analysis of biomedical image sequences (FAMIS).


METHODS
Streptavidin-fluorescein isothiocyanate (FITC), -europium (Eu), -phycoerythrin (PE) and -Texas Red (TR) were chosen to reveal the binding of biotinylated annexin V on apoptotic U937 human leukemic cells and ECV-304 human endothelial cells induced under treatment with 7-ketocholesterol or 7 beta-hydroxycholesterol. Excitation of each fluorochrome was obtained by selection of specific lines (351 + 364 nm, 488 nm) of the argon laser of a confocal microscope. Temporal and spectral series were performed to characterize each fluorochrome. FAMIS was applied to these series to estimate images corresponding to stains.


RESULTS
Each fluorochrome was clearly distinguished, and images showed localization of phosphatidylserine, which was improved by image analysis.


CONCLUSIONS
On apoptotic cells it is possible to analyze differences in the improved visualization of phosphatidylserine in series processed by FAMIS with the use of biotinylated annexin V revealed with streptavidin-FITC, -Eu, -PE or -TR.
Institut National de la Santé et de la Recherche Médicale U494, Centre Hospitalier Universitaire Pitié-Salpêtrière, 75634 Paris, France. kahn@imed.jussieu.fr
E
E
Kahn
G
G
Lizard
F
F
Frouin
J C
JC
Bernengo
C
C
Souchier
G
G
Bessède
O
O
Clément
H
H
Siitari
P
P
Gambert
G
G
Frija
A
A
Todd-Pokropek
D017304:Annexin A5 / Q000378:metabolism*; D017209:Apoptosis; D019921:Biotinylation; D002460:Cell Line; D005063:Europium; D005163:Factor Analysis, Statistical; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal; D010718:Phosphatidylserines / Q000378:metabolism*; D010799:Phycoerythrin; D019809:Streptavidin; D020298:U937 Cells; D014966:Xanthenes
D017304:Annexin A5; D010718:Phosphatidylserines; D014966:Xanthenes; D010799:Phycoerythrin; D005063:Europium; C034657:Texas red; D019809:Streptavidin; D016650:Fluorescein-5-isothiocyanate