Flow cytometric analysis of MCF7 cells, untreated (blue) or treated with Etoposide #2200 (12.5 μM, 24 hr; green) and allowed to recover for 3 days, using SA-β-Gal Fluorescent Substrate (solid lines) compared to unlabeled MCF7 cells (dashed line).
Additional Reagents (Not Supplied):
NOTE: Suggested dilutions and volumes are provided for reference. Optimal conditions for SA-beta-Gal Fluorescent Substrate performance may vary by cell type and container type.
NOTE: Cell treatments to induce senescence should be completed before initiating staining with this kit. Ensure that an untreated control is included, to provide a baseline measurement of fluorescence for comparison.
NOTE: Formaldehyde fixation may be performed at the completion of the incubation step with the SA-beta-Gal Fluorescent Substrate #38154. Fixation with up to 4% formaldehyde for up to 15 minutes will not significantly reduce the fluorescence of the substrate. However, permeabilization with methanol or Triton X-100 will cause a significant reduction in fluorescence. If permeabilization is required for immunolabeling of intracellular targets, conduct an experiment to determine whether this will significantly compromise detection of the fluorescent substrate in that cell model.
posted April 2010
Protocol Id: 1986
The Senescence β-Galactosidase Activity Assay Kit (Fluorescence, Flow Cytometry) allows the quantitative measure of senescence-associated β-galactosidase activity in living cells via flow cytometry using a cell permeable fluorogenic substrate, which becomes fluorescent upon hydrolysis by β-galactosidase. Each kit contains sufficient quantities of reagents to perform up to 100 assays.
Limited capacity to replicate is a defining characteristic of most normal cells and culminates in senescence, an arrested state in which the cell remains viable (1). Senescent cells are not stimulated to divide by serum or passage in culture, and senescence invokes a specific cell cycle profile that differs from most damage-induced arrest processes or contact inhibition (2). An enlarged cell size, expression of pH-dependent β-galactosidase activity (3), and an altered pattern of gene expression (4,5) further characterize senescent cells.
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