- D.N.Y. Fan
- C.A. Schmitt
- Methods in Molecular Biology
- Methods Mol Biol 1896: 93-105
A cell's genomic integrity is at risk when DNA-damaging stress, evoked by mitogenic oncogenes or genotoxic treatment modalities such as radiation or chemotherapy, apply. If the DNA repair machinery fails to fix the damaged site during a temporary cell-cycle arrest, or if massive genotoxic stress overwhelmed the repair capacity, cellular failsafe programs such as apoptosis or senescence will be triggered to limit aberrant propagation of these damaged and potentially harmful cells. After decades of scientific focusing on apoptosis, cellular senescence is increasingly recognized as an equally important but biologically and fundamentally different type of ultimate cell-cycle exit program, because of its lastingly persistent nature and cell-intrinsic and extrinsic roles within the tissue and tumor microenvironment. We established primary apoptosis-compromised, Bcl2-expressing Eμ-myc transgenic mouse lymphomas as a versatile and clinically relevant model system to study therapy-induced senescence (TIS). Given the lack of a single specific senescence-defining marker, we previously exploited co-staining of senescence-associated β-galactosidase (SA-β-gal) activity with immunohistochemical detection of trimethylated histone H3 lysine 9 (H3K9me3), an established S-phase gene expression-controlling, repressive chromatin mark, and the proliferation marker Ki67. This biomarker panel is instrumental to characterize cells as senescent via their high SA-β-gal activity, strong nuclear H3K9me3 expression and Ki67-negative profile. In this chapter, we demonstrate the detection of viable senescent cells by novel methods based on a fluorescent version of the SA-β-gal (fSA-β-gal) assay, combined with immuno-fluoroscence staining of H3K9me3 or Ki67, or analysis of the DNA replication status by incorporating 5-ethynyl-2'-deoxyuridine (EdU) detection into the protocol. Notably, while most senescence markers, irrespective of their specificity and sensitivity, may only be assessed in endpoint assays, we would like to emphasize here the strength of viable fSA-β-gal to track single-cell fate in senescent populations over time.