Senescence Detection Kit - Biovision

Reporter gene assay β-galactosidase substrates - mouse cardiac fibroblasts

Experiment
Reporter gene assay β-galactosidase substrates - mouse cardiac fibroblasts
Product
Senescence Detection Kit - Biovision from Biovision
Manufacturer
Biovision

Protocol tips

Upstream tips
Pretreat cells with TM5441 for 24 h followed by Doxorubicin (50 nM) treatment in triplicate for 4 days.
Protocol tips
Fix cells with fixative solution for 15 min at room temperature.

Add 0.5 ml of the Staining Solution Mix to each well. Cover the plate. Incubate overnight at 37°C.

Publication protocol

The widely used biomarker for senescent cells is the SA-β-galactosidase activity. SA-β-gal activity is detectable at pH 6.0 in senescent cells (BioVision, Milpitas, CA). Beta-galactosidase activity is expressed from GLB1, the gene encoding lysosomal-galactosidase. Cardiomyocytes H9c2, endothelial cells EA.hy926, mouse embryonic fibroblasts (MEFs) and neonatal mouse cardiac fibroblasts (nMCFs) were cultured in 12-well culture plates and pretreated with TM5441 for 24 h followed by Doxorubicin (50 nM) treatment in triplicate for 4 days. At the end of treatment, cells were processed for SA-β-gal using Senescence Assay Kit (Catalog Cat#K320-250; BioVision, Milpitas, CA). In brief, culture medium was removed and cells were washed once with 1X PBS and fixed with fixative solution for 15 min at room temperature. After fixation, cells were washed twice with 1XPBS and staining solution containing staining supplement and X-gal was added to each well and mixed. Plates were covered with aluminum foil and incubated overnight at 37°C without CO2. The SA-β-gal staining in cells were observed under a microscope for development of blue color and photographed

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Papers

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Paper title
A small molecule inhibitor of PAI-1 protects against doxorubicin-induced cellular senescence
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Manufacturer protocol

Download the product protocol from Biovision for Senescence Detection Kit - Biovision below.

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