Abstract
Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.
Highlights
Normal somatic cells, with the exception of germ cells and some stem cells, display a finite replicative capacity
We report here the development of a novel method to stain and observe total protein by high resolution fluorescence microscopy, which we apply to study protein content changes in senescing human and mouse fibroblasts in vitro, and in young and aged mouse liver tissue in vivo
Total protein content of early passage and senescent fibroblasts in culture We first confirmed previous reports that senescent cells contain more protein using the strain of normal human diploid fibroblasts (HDF) commonly used in our lab (LF1, see Methods)
Summary
With the exception of germ cells and some stem cells, display a finite replicative capacity. This phenomenon was first described in cultured human fibroblasts [1], and is commonly referred to as replicative cellular senescence. These observations were extended to a wide variety of vertebrate species and cell types, and senescence is believed to be a general property of replicative cells [2]. Complex, and often cell-type specific nature of these changes this remains an active area of investigation
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