Robust Multiparametric Assessment of Cellular Senescence

Cellular senescence, the irreversible loss of replicative capacity, is both a tumor suppressor mechanism and a contributor to the age-related loss of tissue function. However, the role of cellular senescence in vivo has been unclear, mostly because of the absence of cellular markers specific enough to identify the state (senescent or proliferating) of individual cells in tissues. Recently, we have tested the robustness of multiple senescence candidate markers by comparing them to a dynamic stimulation model, which estimates the fraction of senescent cells with high precision. We found that the absence of the proliferation markers Ki67 and PCNA combined with high density DNA damage foci (>5 γH2AX foci per nucleus) was the best quantitative indicator of cellular senescence. In this chapter, we describe protocols for the dual immunofluorescence-based quantification of Ki67/PCNA and γH2AX in both fixed cells and paraffin-embedded tissues.