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The CellEvent Senescence Green Flow Cytometry Assay Kit enables flow cytometric detection of cellular senescence via β-galactosidase hydrolysis. The kit contains an optimized buffer and a fluorescein-based reagent that contains two galactoside moieties, making it a specific target for β-galactosidase. Activation of β-galactosidase is commonly used as a biomarker for senescent cells. This hydrolase enzyme resides in lysosomes and converts β-galactosides into monosaccharides under acidic pH conditions. The enzyme-cleaved product is retained within the cell due to covalent binding of intracellular proteins and emits a fluorogenic signal that has absorption/emission maxima of 490/514 nm.
CellEvent Senescence Green Flow Cytometry Assay Kit features include:
• Fluorescent detection of β-galactosidase hydrolysis by flow cytometry
• Fixed cell assay
• Multi-color compatibility
When normal cells reach the end of their limited replicative lifespan, commonly referred to as the Hayflick limit, they enter into a senescent phase where they remain metabolically active without undergoing cell death processes. These senescent cells adopt a specific phenotypic state that includes the appearance of multi-nucleated cells, increased vacuolization, expression of pH-dependent β-galactosidase, and morphological changes where cells become enlarged and extended. Through a variety of mechanisms, they may play a role in tumor suppression, tumor progression, aging, and tissue repair.
β-galactosidase resides in lysosomes and converts β-galactosides into monosaccharides under acidic pH conditions. The activity is optimal under lysosomal pH 4, but conventional assays measure at pH 6. It has been shown that normalized β-galactosidase activity is twice as high in senescent cells as in presenescent cells regardless of the pH value used for testing.
Traditionally, the colorimetric substrate for β-galactosidase, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (x-gal), has long been used to detect metabolic activity in cells in vitro. Upon hydrolysis by β-galactosidase, x-gal converts to a blue precipitate, which is not suitable for flow cytometric assays. Therefore, we have developed a sensitive, fluorescent substrate for β-galactosidase that can be used in flow cytometry assays for the detection of senescent cells.