6th ESACP Congress, Heidelberg, April 7-11, 1999

A076
CONFOCAL ANALYSIS OF DUAL COLOR STAINING OF MECHANICAL MEDIATORS OF CELL MOTILITY.
Kahn E 1, Siméon J 2, Knoll D 2, Frouin F 1, Di Paola R 1, Lelièvre JC 2

1) Inserm U494, CHU Pitié-Salpétrière, Paris, France 2) Université Paris 6 and LBHP CNRS ESA 7057, CHU Pitié Salpétrière, Paris, France

PURPOSE: To show that cellular preparations requiring characterization of mechanical mediators of cell motility (deformation, adhesion) can be analyzed by confocal microscopy and image processing, in order to get 3D localization of the proteins corresponding to these mediators : actin and integrin alphaVbeta3. METHODS: We used tetramethyl rhodamine isothyocianate (TRITC) to stain actin and fluorescein isothyocianate (FITC) to stain integrin of HL60 cells. These cells can be processed to express their motility (differentiation and activation). Excitation of each fluorochrome was obtained by selection of specific bands (488nm, 514nm) of the argon laser of a confocal microscope. Temporal and spectral series were also performed to characterize each fluorochrome. Z-series were performed when required to define the best focalizations and localize both mediators. Factor analysis of medical images (FAMIS) was applied to these series to estimate images corresponding to stains and specific focal images, see reference. RESULTS: Each fluorochrome was clearly distinguished and images showed localizations of proteins. 3D analysis improved characterization of proteins. Actin was clearly located in the front of the moving cell, while integrin was located at the rear. CONCLUSIONS: It is possible to analyze differences in the focus and to improve their visualization on selected planes by FAMIS. It is possible to study localization of cellular proteins via 3D multicolor experiments. It is possible to study mechanical mediators in numerous experimental conditions of motility assays
REFERENCE: E. Kahn et al, 1997, Cytometry 28:269-279