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Open Access Highly Accessed Methodology article

Quantitative digital in situ senescence-associated β-galactosidase assay

Liran I Shlush123, Shalev Itzkovitz4, Ariel Cohen4, Aviad Rutenberg1, Ron Berkovitz12, Shiran Yehezkel1, Hofit Shahar12, Sara Selig13* and Karl Skorecki13

Author Affiliations

1 Laboratory of Molecular Medicine, Rappaport Faculty of Medicine and Research Institute, Technion, Haifa 31096, Israel

2 Israeli Naval Medical Institute, Haifa, Israel

3 Rambam Health Care Center, POB 9602, Haifa 31096, Israel

4 Department of Computer Science and Applied Mathematics, Weizmann Institute of Science, Rehovot 76100 Israel

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BMC Cell Biology 2011, 12:16  doi:10.1186/1471-2121-12-16

Published: 15 April 2011

Abstract

Background

Cellular senescence plays important roles in the aging process of complex organisms, in tumor suppression and in response to stress. Several markers can be used to identify senescent cells, of which the most widely used is the senescence-associated β-galactosidase (SABG) activity. The main advantage of SABG activity over other markers is the simplicity of the detection assay and the capacity to identify in situ a senescent cell in a heterogeneous cell population. Several approaches have been introduced to render the SABG assay quantitative. However none of these approaches to date has proven particularly amenable to quantitative analysis of SABG activity in situ. Furthermore the role of cellular senescence (CS) in vivo remains unclear mainly due to the ambiguity of current cellular markers in identifying CS of individual cells in tissues.

Results

In the current study we applied a digital image analysis technique to the staining generated using the original SABG assay, and demonstrate that this analysis is highly reproducible and sensitive to subtle differences in staining intensities resulting from diverse cellular senescence pathways in culture. We have further validated our method on mouse kidney samples with and without diabetes mellitus, and show that a more accurate quantitative SABG activity with a wider range of values can be achieved at a pH lower than that used in the conventional SABG assay.

Conclusions

We conclude that quantitative in situ SABG assay, is feasible and reproducible and that the pH at which the reaction is performed should be tailored and chosen, depending on the research question and experimental system of interest.