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Analysis of Cellular Senescence in Culture In Vivo: The Senescence‐Associated β‐Galactosidase Assay

Debdutta Bandyopadhyay1,  Catherine Gatza1,  Lawrence A. Donehower1,  Estela E. Medrano1

1Baylor College of Medicine, Houston, Texas

Publication Name: 
Current Protocols in Cell Biology
Unit Number: 
Unit 18.9
DOI: 
10.1002/0471143030.cb1809s27
Online Posting Date: 
July, 2005
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Abstract

Replicative senescence, a process first described almost 40 years ago, entails irreversible growth arrest with sustained metabolic functions, and it is also associated with increased resistance to apoptotic signals. Interest in this process has increased greatly over the last 10 years, as it has been demonstrated that senescence can function as a potent tumor suppressor mechanism. Although mounting evidence suggests that the senescent phenotype is associated with an extraordinarily complex array of gene expression patterns and interactions with the microenvironment, there is only one widely accepted marker for distinguishing such cells in vitro and in vivo. This marker is the senescence-associated expression of a pH 6 -galactosidase (SA--gal). Here, a method for analyzing SA--gal expression in cultured cells and in human and animal tissues is described, and important parameters to consider when performing such assays are also highlighted.

Keywords: senescence-associated -galactosidase assay; cultured cells; animal tissues; senescence

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Assessment of Cellular Senescence in Culture Using the Senescence-Associated -Galactosidase Assay
  • Alternate Protocol: Assessment of Cellular Senescence in Murine Tissue Using the Senescence-Associated -Galactosidase Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol: Assessment of Cellular Senescence in Culture Using the Senescence-Associated -Galactosidase Assay

 Materials
  • Cells to be analyzed
  • 1× PBS (appendix 2A)
  • Fixing solution (see recipe)
  • Staining solution (see recipe)
  • Mounting medium
  • Colorless nail polish
  • Coverslips, sterile
  • 10-cm-diameter petri dishes
  • Platform or orbital shaker
  • Light microscope
  • Glass microscope slides

Alternate Protocol: Assessment of Cellular Senescence in Murine Tissue Using the Senescence-Associated -Galactosidase Assay

 Additional Materials (also see Basic Protocol)
  • Tissue of interest (e.g., liver, spleen, kidney)
  • Optimum cutting temperature (OCT) compound
  • Liquid nitrogen
  • 0.5% (v/v) glutaraldehyde (see recipe)
  • Senescence-associated -galactosidase stain (see recipe)
  • Hematoxylin (Sigma) Mounting medium
  • Eosin (Sigma)
  • Cryomold
  • Standard cryotome (Thermo Electron Anatomical Pathology or equivalent)
  • Positively charged glass slides (e.g., VWR)
  • Humidity chamber: plastic box with lid
  • Pap pen
  • Coplin jars
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 18.9.1
    Senescence-associated -galactosidase staining in normal human melanocytes (as performed in Basic Protocol). Senescent cells exhibit strong perinuclear activity. Note the enlarged, flat morphology of the senescent melanocytes compared with the much smaller, spindle-shaped morphology of the proliferating melanocytes. Similar morphological changes are also observed in other senescent cell types.

    View Image
  • Figure 18.9.2
    (A) Senescence-associated -galactosidase staining in murine liver (performed as in Alternate Protocol). Senescent cells exhibit blue perinuclear staining (arrow) upon exposure to Xgal at pH 6.0, whereas nonsenescent cells do not. Nuclei are stained with hematoxylin. (B) Analysis of senescence in young (3-month-old) and old (21-month-old) wild-type murine liver tissue. 6-µm sections of young and old liver were treated with the senescence-associated -galactosidase stain as described in the Alternate Protocol. Sections were observed under light microscopy to determine the percentage of senescent cells present. Levels of senescence were elevated in old wild-type liver compared with young wild-type liver (n = 4 for both sample sets).

    View Image

Literature Cited

Literature Cited
    Bringold, F. and Serrano, M. 2000. Tumor suppressors and oncogenes in cellular senescence. Exp. Gerontol. 35:317-329.
    Busuttil, R.A., Rubio, M., Dolle, M.E., Campisi, J., and Vijg, J. 2003. Oxygen accelerates the accumulation of mutations during the senescence and immortalization of murine cells in culture. Aging Cell 2:287-294.
    Campisi, J. 2003. Cellular senescence and apoptosis: How cellular responses might influence aging phenotypes. Exp. Gerontol. 38:5-11.
    Cao, L., Li, W., Kim, S., Brodie, S.G., and Deng, C.X. 2003. Senescence, aging, and malignant transformation mediated by p53 in mice lacking the Brca1 full-length isoform. Genes Dev. 17:201-213.
    Choi, J., Shendrik, I., Peacocke, M., Peehl, D., Buttyan, R., Ikeguchi, E.F., Katz, A.E., and Benson, M.C. 2000. Expression of senescence-associated beta-galactosidase in enlarged prostates from men with benign prostatic hyperplasia. Urology 56:160-166.
    Dimri, G.P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E.E., Linskens, M., Rubelj, I., and Pereira-Smith, O. 1995. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. U.S.A. 92:9363-9367.
    Hayflick, L. 1965. The limited in vitro lifespan of human diploid cell strains. Exp. Cell Res. 25:585-621.
    Kim, H., You, S., Farris, J., Kong, B.W., Christman, S.A., Foster, L.K., and Foster, D.N. 2002. Expression profiles of p53-, p16(INK4a)-, and telomere-regulating genes in replicative senescent primary human, mouse, and chicken fibroblast cells. Exp. Cell Res. 272:199-208.
    Kurz, D.J., Decary, S., Hong, Y., and Erusalimsky, J.D. 2000. Senescence-associated -galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells. J. Cell Sci. 113:3613-3622.
    Mishima, K., Handa, J.T., Aotaki-Keen, A., Lutty, G.A., Morse, L.S., and Hjelmeland, L.M. 1999. Senescence-associated -galactosidase histochemistry for the primate eye. Invest. Ophthalmol. Vis. Sci. 40:1590-1593.
    Paradis, V., Youssef, N., Dargere, D., Ba, N., Bonvoust, F., Deschatrette, J., and Bedossa, P. 2001. Replicative senescence in normal liver, chronic hepatitis C, and hepatocellular carcinomas. Hum. Pathol. 32:327-332.
    Price, J.S., Waters, J.G., Darrah, C., Pennington, C., Edwards, D.R., Donell, S.T., and Clark, I.M. 2002. The role of chondrocyte senescence in osteoarthritis. Aging Cell 1:57-65.
     
 
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