Cellular Identification and Quantification of Senescence-Associated β-Galactosidase Activity In Vivo

  • Bennett G. Childs
  • Tyler J. Bussian
  • Darren J. BakerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1896)


Senescence-associated β-galactosidase (hereafter SA-β-gal) staining has now been employed for more than 20 years to identify the presence of senescent cells (Dimri et al., Proc Natl Acad Sci U S A 92:9363–9367, 1995). These cells, characterized by a permanent cell-cycle arrest (Hayflick and Moorhead, Exp Cell Res 25:585–621, 1961) and the production of a distinct secretory phenotype of cytokines, chemokines, and proteases (Coppe et al., PLoS Biol 6:2853–2868, 2008), have received much attention in recent years for their impacts on diverse biological processes. Here we describe a method to identify and quantify the specific cells that become senescent in vivo using transmission electron microscopy after SA-β-gal staining that can be used in countless scenarios.

Key words

Cellular senescence Transmission electron microscopy Senescence-associated β-galactosidase 



This work was supported by the National Institutes of Health (R01AG053229), the Glenn Foundation for Medical Research, the Ellison Medical Foundation, and the Mayo Clinic Children’s Research Center to D.J.B.


  1. 1.
    Dimri G, Lee X, Basile G et al (1995) A biomarker that identifies senescent human cells in culture and aging skin in vivo. Proc Natl Acad Sci U S A 92:9363–9367CrossRefGoogle Scholar
  2. 2.
    Hayflick L, Moorhead P (1961) The serial cultivation of human diploid cell strains. Exp Cell Res 25:585–621CrossRefGoogle Scholar
  3. 3.
    Coppe J, Patil C, Rodier F et al (2008) Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic Ras and the p53 tumor suppressor. PLoS Biol 6:2853–2868CrossRefGoogle Scholar
  4. 4.
    Storer M, Mas A, Robert-Moreno A et al (2013) Senescence is a developmental mechanism that contributes to embryonic growth and patterning. Cell 155:1119–1130CrossRefGoogle Scholar
  5. 5.
    Munoz-Espin D, Canamero M, Maraver A et al (2013) Programmed cell senescence during mammalian embryonic development. Cell 155:1104–1118CrossRefGoogle Scholar
  6. 6.
    Ritschka B, Storer M, Mas A et al (2017) The senescence-associated secretory phenotype induces cellular plasticity and tissue regeneration. Genes Dev 31:172–183CrossRefGoogle Scholar
  7. 7.
    Chiche A, Le Roux I, von Joest M et al (2017) Injury-induced senescence enables in vivo reprogramming in skeletal muscle. Cell Stem Cell 20:407–414CrossRefGoogle Scholar
  8. 8.
    Demaria M, Ohtani N, Youssef S et al (2014) An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA. Dev Cell 31:722–733CrossRefGoogle Scholar
  9. 9.
    Baker D, Childs B, Durik M et al (2016) Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan. Nature 530:184–189CrossRefGoogle Scholar
  10. 10.
    Baker D, Wijshake T, Tchkonia T et al (2011) Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 479:232–236CrossRefGoogle Scholar
  11. 11.
    Jeon O, Kim C, Laberge R et al (2017) Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment. Nat Med 23:775–781CrossRefGoogle Scholar
  12. 12.
    Childs B, Baker D, Wijshake T et al (2016) Senescent intimal foam cells are deleterious at all stages of atherosclerosis. Science 354:472–477CrossRefGoogle Scholar
  13. 13.
    Stollewerk A, Klamby C, Cantera R (1996) Electron microscopic analysis of Drosophila midline glia during embryogenesis and larval development using beta-galactosidase expression as endogenous cell marker. Microsc Res Tech 35:294–306CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Bennett G. Childs
    • 1
  • Tyler J. Bussian
    • 1
  • Darren J. Baker
    • 1
    • 2
    Email author
  1. 1.Department of Biochemistry and Molecular BiologyMayo ClinicRochesterUSA
  2. 2.Department of Pediatric and Adolescent MedicineMayo ClinicRochesterUSA

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