Combination of quantification and observation methods for study of "Side Population" cells in their "in vitro" microenvironment.
BACKGROUND
Qualitative and quantitative analyses of the rare phenotypic variants in in vitro culture systems is necessary for the understanding of cell differentiation in cell culture of primary cells or cell lines. Slide-based cytometry combines image acquisition and data treatment, and associates the power of flow cytometry (FCM) and the resolution of the microscopic studies making it suitable for the analysis of cells with rare phenotype. In this paper we develop a method that applies these principles to a particularly hot problem in cell biology, the study of stem cell like cells in cultures of primary cells, cancer cells, and various cell lines.


METHODS
The adherent cells were labeled by the fluorescent dye Hoechst 33342. The images of cell populations were collected by a two-photon microscope and processed by a software developed by us. The software allows the automated segmentation of the nuclei in a very dense cell environment, the measurement of the fluorescence intensity of each nucleus and the recording of their position in the plate. The cells with a given fluorescence intensity can then be located easily on the recorded image of the culture plate for further analysis.


RESULTS
The potential of our method is illustrated by the identification and localization of SP cells in the cultures of the C2C12 cell line. Although these cells represent only about 1% of the total population as calculated by flow cytometry, they can be identified in the culture plate with high precision by microscopy.


CONCLUSIONS
Cells with the rare stem-cell like phenotype can be efficiently identified in the undisturbed cultures. Since the fluorescence intensity of rare events and the position of thousands of surrounding cells are recorded at the same time, the method associates the advantage of the FCM analysis and the microscopic observation.
Benchaouir
Rachid
R
GENETHON - Centre National de la Recherche Scientifique UMR 8115, 1 bis, rue de l'Internationale 91002 Evry, France.
Picot
Julien
J
Greppo
Nicolas
N
Rameau
Philippe
P
Stockholm
Daniel
D
Garcia
Luis
L
Paldi
Andras
A
Laplace-Builhé
Corinne
C
D000818:Animals; D001562:Benzimidazoles; D018929:Cell Culture Techniques; D002469:Cell Separation; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods; D005456:Fluorescent Dyes; D019044:Image Cytometry; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D032446:Myoblasts / Q000378:metabolism; D013234:Stem Cells / Q000145:classification* / Q000166:cytology
D001562:Benzimidazoles; D005456:Fluorescent Dyes; C017807:bisbenzimide ethoxide trihydrochloride