Protocols to detect senescence-associated beta-galactosidase (SA-βgal) activity, a biomarker of senescent cells in culture and in vivo

Abstract

Normal cells can permanently lose the ability to proliferate when challenged by potentially oncogenic stress, a process termed cellular senescence. Senescence-associated beta-galactosidase (SA-betagal) activity, detectable at pH 6.0, permits the identification of senescent cells in culture and mammalian tissues. Here we describe first a cytochemical protocol suitable for the histochemical detection of individual senescent cells both in culture and tissue biopsies. The second method is based on the alkalinization of lysosomes, followed by the use of 5-dodecanoylaminofluorescein di-beta-D-galactopyranoside (C12FDG), a fluorogenic substrate for betagal activity. The cytochemical method takes about 30 min to execute, and several hours to a day to develop and score. The fluorescence methods take between 4 and 8 h to execute and can be scored in a single day. The cytochemical method is applicable to tissue sections and requires simple reagents and equipment. The fluorescence-based methods have the advantages of being more quantitative and sensitive.

A Pharmacological Chaperone Molecule Induces Cancer Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters

“…69 Briefly, compound-treated MCF7 cells were fixed in 2% formaldehyde and 0.2% Caspase assay. MCF7 cells in a 6-well plate were treated with GTC365 for 48 h. The caspase-3/7 assay was conducted using DEVD-AFC as a substrate of capase-3/7.…”

Ex vivo observation of human intervertebral disc tissue and cells isolated from degenerated intervertebral discs

“…an intracellular green-blue, insoluble precipitate, using the Senescence Detection Kit b-Gal (Calbiochem, QIA117) [11].…”

EGF promotes mammalian cell growth by suppressing cellular senescence

“…Upon 3 days of culture in the presence of erlotinib, HME and HBE cells appeared morphologically similar to those deprived of EGF, failed to proliferate, and contained high SA-β-gal activity ( Figure 1D and 1E, Supplementary information, Figure S1F and S1G), indicating that EGF suppresses senescence by activating EGFR in these cell types. To quantify the level of senescence induced by EGFR inhibition, we made use of C 12 FDG, a substrate that becomes fluorescent after SA-β-gal-catalyzed cleavage [3]. Incubation with C 12 FDG and subsequent analysis by flow cytometry revealed that ~70% of HME and HBE cells were senescent after two days of treatment with 1 µM erlotinib ( Figure 1F-1H and Supplementary information, Figure S1H-S1J).…”