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.
Bibliographical noteFunding Information:
We thank Maty Tzukerman for her helpful discussions and suggestions, Woodring Wright for providing us with BJ cells and Shiri Kalet-Litman for her help in the experiments with the diabetic mice. We are grateful to Sagi Abelson and Annie Rebibo-Sabbah for their technical help and to Ayala Ofir for her help with the confocal microscopy. This research was supported by the Israel Science Foundation (grant No. 542/05) (S.S.), the Deborah Grant (grant No 2007646) (K.S., L.S., S.S.), the Arthur and Rosalinde Gilbert Estate grants and the Sidney Kremer Kidney research Fund at the Technion (K.S).