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6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A076
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
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
REFERENCE: E. Kahn et al, 1997, Cytometry 28:269-279