Cell death, cell cycle arrest and cellular senescence are three distinct cellular responses that can be induced by oncogene activation and diverse anti-cancer agents, and this often requires the action of the tumour suppressor TP53. Within a cell population, or even within an individual cell, these processes are not necessarily mutually exclusive. It is therefore important to measure all these processes simultaneously. However, current assays generally visualise only one or at best two responses, often only detecting the dominant one. Here, we present a novel flow cytometric assay that allows simultaneous assessment of cell viability and cell cycling through measurement of DNA content and DNA synthesis, and markers of cell senescence at the single cell level. We demonstrate that this assay can be performed on both human and murine cells, that are either cancerous or non-transformed, and can help to dissect complex cell fate decisions. We believe that this experimental tool will be useful for the study of diverse biological processes. 

Flow cytometric single cell-based assay to simultaneously detect cell death, cell cycling, DNA content and cell senescence

FACS-Gal is a system that enables the fluorescence activated cell sorter (FACS) to sensitively assay expression of the E. Coli lacZ (β-galactosidase, β-gal) reporter gene, and sort viable cells based on the levels of expression of this enzyme.1,2 The system depends on the enzymatic conversion of the nonfluorescent β-gal substrate fluorescein di-β-galactopyranoside (FDG), into the fluorescent molecule fluorescein. FACS-Gal effectively combines a selectable marker with a reporter gene and in the combination produces novel experimental possibilities. The system is an approach to applying the analytical and sorting capabilities of the FACS to solving problems in molecular biology. FACS-Gal and its variants have been used with cells from a variety of organisms including E. Coli,3 yeast,3 Drosophila,4 transgenic mice,5–7 and most frequently with mammalian cell lines.8–13 For a more extensive list of references using the FACS-Gal assay see.14 This review focuses on the technical basics of FACS-Gal, how to most productively use the system, and the strengths and weaknesses of this system in comparison to other presently available alternatives.

FACS-Gal: Flow Cytometric Assay of β-galactosidase in Viable Cells

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FACS-Gal: Flow Cytometric Assay of β-galactosidase in Viable Cells