title: Quantitative digital in situ senescence-associated β-galactosidase assay.
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.
authors.affiliation: Laboratory of Molecular Medicine, Rappaport Faculty of Medicine and Research Institute, Technion, Haifa, Israel.
authors.forename: Liran I
authors.initials: LI
authors.lastname: Shlush
authors.forename: Shalev
authors.initials: S
authors.lastname: Itzkovitz
authors.forename: Ariel
authors.initials: A
authors.lastname: Cohen
authors.forename: Aviad
authors.initials: A
authors.lastname: Rutenberg
authors.forename: Ron
authors.initials: R
authors.lastname: Berkovitz
authors.forename: Shiran
authors.initials: S
authors.lastname: Yehezkel
authors.forename: Hofit
authors.initials: H
authors.lastname: Shahar
authors.forename: Sara
authors.initials: S
authors.lastname: Selig
authors.forename: Karl
authors.initials: K
authors.lastname: Skorecki
mesh_terms: D000818:Animals; D016922:Cellular Senescence; D057075:Enzyme Assays / Q000379:methods*; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D012815:Signal Processing, Computer-Assisted; D001616:beta-Galactosidase / Q000378:metabolism*
chemical_list: D006861:Hydrogen Peroxide; D001616:beta-Galactosidase