Cytometry A. 2007 Apr;71(4):251-7. doi: 10.1002/cyto.a.20367.

Combination of quantification and observation methods for study of "Side Population" cells in their "in vitro" microenvironment.

Cytometry. Part A : the journal of the International Society for Analytical Cytology

Rachid Benchaouir, Julien Picot, Nicolas Greppo, Philippe Rameau, Daniel Stockholm, Luis Garcia, Andras Paldi, Corinne Laplace-Builhé

PMID: 17279573 DOI: 10.1002/cyto.a.20367

Abstract

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.

CONCLUSION: 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.

(c) 2007 International Society for Analytical Cytology.

Substances

• Benzimidazoles
• Fluorescent Dyes
• bisbenzimide ethoxide trihydrochloride

MeSH terms

• Animals
• Benzimidazoles
• Cell Culture Techniques
• Cell Separation
• Cells, Cultured
• Flow Cytometry / instrumentation
• Flow Cytometry / methods
• Fluorescent Dyes
• Image Cytometry
• Mice
• Microscopy, Fluorescence / instrumentation
• Microscopy, Fluorescence / methods
• Myoblasts / metabolism
• Stem Cells / classification
• Stem Cells / cytology

Publication Types

• Evaluation Study
• Journal Article