Fluorescent Method to Detect Senescence-Associated Beta Galactosidase

February 23, 2022

BlueskyReddit
Faculty of Medicine of University of Coimbra
Advanced Therapies Group
Graduate Student

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Company:

Thermo Fisher Scientific

Product Name:

C12FDG (5-Dodecanoylaminofluorescein Di-β-D-Galactopyranoside)

Catalog Number:

D2893

The β-galactosidase substrate, C12FDG has been covalently modified to include a 12-carbon lipophilic moiety. Once inside the cell, the substrate is cleaved by β-galactosidase producing a fluorescent product that is retained by the cells. This is a great alternative to the colorimetric method often used to detect SA-beta Galactosidase. It allows for easy analysis of positive cells. Protocol to use C12FDG to detect SA-beta Galactosidase can be found here: doi 10.1038/nprot.2009.191

Experimental Design and Results Summary

Application

Detect senescence-associated beta Galactosidase

Starting Material

Human cells

Tips

SA-beta Galactosidase optimal working pH is 6. To detect its presence, cells must be incubated with bafylomicin or in the absence of CO2, in order to achieve this pH value. Both methods work well and are detailed in the article with doi 10.1038/nprot.2009.191

Results Summary

To understand if this fluorescence method is as reliable as the most used colorimetric assay, I performed both in parallel. The percentage of cells with senescence-associated beta-galactosidase was very similar with both methods. The fluorescent method has the advantage of being easier to quantify using programs such as ImageJ.

DOI or PMID #

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Summary

The Good

Easy to quantify and to use

The Bad

Does not work in all cell types. It worked well in HUAECs, HUVECs and HCAECs but not in primary fibroblasts or BMECs.

The Bottom Line

This fluorescent method to detect Senescence-associated beta-galactosidase is easier to quantify than the colorimetric method. It also allows cells to remain alive during and after the assay. The only drawback is that cells must be incubated in pH=6 for 2-3h to detect this enzyme. However, it is possible to use in parallel with other fluorescent dyes if needed and if the pH requirement does not affect said dye. Be aware, however, that after the C12FDG assay, cells cannot be fixed without affecting the assay.

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