Lysosomal Profiling With LysoTracker For Quantitative Assessment of Cellular Senescence In Human Fibroblasts.

Cellular senescence is a stable cell-cycle arrest state associated with characteristic phenotypes, including enlarged cell morphology, altered secretory signaling, and pronounced lysosomal remodeling. Senescent cells commonly exhibit expansion of the acidic endo-lysosomal compartment, accompanied by changes in luminal acidity and degradative capacity, creating an opportunity for simple live-cell readouts of senescence-linked organelle remodeling. This work describes a live-cell imaging protocol that uses LysoTracker Deep Red, an acidotropic fluorescent dye, to provide an indirect, pH-dependent proxy for the acidic organelle compartment as a correlate of senescence burden. The method is demonstrated in IMR-90 human lung fibroblasts undergoing replicative senescence across serial passaging. The protocol details cell culture and passage tracking, LysoTracker staining, fluorescence imaging, and image-based quantification of lysosomal signal intensity and signal-positive area per cell. Senescence-associated β-galactosidase (SA-β-Gal) staining on parallel cultures is included as an optional confirmatory marker rather than a reference standard. Representative outcomes show higher acidotropic fluorescent dye signal and larger lyso-positive regions in late-passage cultures than in early-passage controls, consistent with expansion of the acidic organelle compartment during senescence. Because the readout depends on compartment volume, proton gradient, and dye availability, it is best interpreted as an indirect correlate of lysosomal remodeling rather than a direct measure of lysosome number or biogenesis. The protocol is simple to adopt and can be adapted to other cell types or senescence-inducing stresses, providing a practical, quantitative complement to conventional endpoint assays.