Cellular senescence (CS) is a term used to describe the process wherein somatic cells of complex eukaryotic organisms progressively lose replicative capacity. The relationship between CS and organismal aging is still unclear although recent studies in non-human and human primates have strongly implicated a correlation between organismal and cellular aging [1, 2]. Overall, recent studies have suggested that CS is a final common pathway resulting from activation of the cellular DNA damage response (DDR) by various stressors that converge on the p53 and/or pRB pathways. Different DDR inducing stimuli can lead to various types of CS. Among those most thoroughly investigated is the activation of DDR by telomere attrition which leads to cell cycle arrest termed replicative senescence (RS) or telomere-initiated CS [3–5]. Other well studied forms of CS include oncogene-induced senescence [6–8], cell structure induced senescence related to dysfunctional Lamin A , and stress-induced premature senescence (SIPS), the latter most thoroughly studied in relation to oxidative stress [10–12]. These various triggers of CS might not necessarily be mutually exclusive. Furthermore, DDR might not be the exclusive mechanism for triggering CS as protein damage, epigenetic changes  and additional processes have also been implicated [5, 14]. In complex long-lived organisms CS is considered to be a tumor suppressor mechanism similar to apoptosis and autophagy . However, in contrast to apoptosis and autophagy, which are irreversible and lead to cell death, senescent cells maintain partial metabolic functionality without dividing, and have been shown to have the capacity to revert back to a proliferative state .
Several markers of senescence have been described . Among others these include G1 cell cycle arrest detected by lack of DNA replication, cytological markers such as senescence-associated heterochromatin foci (SAHF), senescence-associated DNA-damage foci, as well as cell structure changes such as cell size and lysosomal β-galactosidase activity detected at pH 6.0 defined as senescence-associated β-galactosidase (SABG) activity [16, 17]. Since first reported, SABG activity has been the most extensively utilized biomarker for CS both in in situ[16, 18–20] and in in vitro studies (reviewed in ). In many studies the identification of cells as being senescent rests solely on the SABG assay. The popularity of this method can be attributed to its simplicity and apparent specificity for CS regardless of the initiating trigger, as well as the ability to visualize senescent cells in a heterogeneous population .
Despite the extensive utilization of the SABG assay for CS determination, the origin of SABG activity and its role in CS were unknown for several years following its initial description. A number of studies have proposed that lysosomal β-galactosidase activity increases in senescent cell up to a degree that surpasses a threshold level that renders the activity detectable at a suboptimal pH 6.0 [21, 22]. A later study clearly demonstrated that the SABG activity arises from the lysosomal β-galactosidase 1 (GLB1) gene product . In senescence cells, both the mRNA and the protein levels of this gene are significantly elevated, and the enzymatic activity increases concomitantly . Furthermore, the enhanced enzymatic activity in senescence can be measured both at the optimal pH for activity - pH 4.5 as well as at the suboptimal pH 6.0. These findings demonstrate that the significantly increased SABG activity at senescence is the basis for the activity detected at the suboptimal pH 6.0, and therefore used as a marker for senescence .
The extent of the senescence-induced increase in lysosomal β-galactosidase can be measured by Western blotting or soluble enzymatic activity , however the activity of β-galactosidase is most easily and robustly detected by histochemical staining with X-gal serving as a substrate . In addition to age related accumulation of lysosomal β-galactosidase in senescent cells probably due to the increased lysosomal content in the cell, other yet unknown factors such as functional differences in senescent lysosomes may contribute to the very high levels of β-galactosidase observed by SABG staining at pH 6.0 .
Despite its ease and utility, concerns have arisen regarding the specificity and reproducibility of the SABG assay. Studies that question the specificity of the SABG assay as a CS marker have found SABG activity in quiescent cell cultures , confluent non-transformed fibroblasts cultures [25, 26] and in serum starved cells . Other concerns relate to the nature of the semi-quantitative measure. Previously described scoring has been based on experimentalist-dependent determination of a given cell as being positively or negatively stained, and may not be anchored in reproducible criteria. Furthermore, the intensity of blue staining in positive cells may be difficult to quantify, such that cells with strong, moderate, or weak blue staining may all be recognized as equally positive. This renders the method insensitive to subtle effects of various stressors on CS, and might contribute to the inconsistency in replicating SABG assay results in skin biopsies .
The foregoing motivated us to develop a quantitative in situ SABG assay, which could be more easily applicable and reproducible in the study of CS both in vitro and in situ. We have utilized the framework of the widely utilized protocol of the in situ SABG assay , and applied digital-image processing in order to perform quantification of the assay staining. In addition, we have also varied the pH of the assay to broaden the range of histochemically detectable activity. We show validation of this quantitative in situ SABG assay on cultured human foreskin fibroblasts and frozen kidney biopsies from normal and diabetic mice, stained under different assay conditions. The values derived from this analysis are termed β-galactosidase activity values (BGAVs) and are highly sensitive and reproducible.