Abstract
Senescence-associated β-galactosidase (SAβ-gal) is a convenient histological technique used to identify senescent cells. Its ease of use is helpful to initially screen and detect senescent cells in heterogeneous cell populations both in vitro and in vivo. However, SAβ-gal staining is not an unequivocal marker of the senescent state, and diagnosis of such usually requires additional markers demonstrating an absence of proliferation and expression of cell-cycle inhibitors. Nonetheless, SAβ-gal remains one of the most widely used biomarkers of senescent cells. Recently, by measuring SAβ-gal activity, the expression of the cyclin-dependent kinase inhibitor p21 (waf1/cip1) and demonstrating a lack of proliferation, we identified senescent cells in the developing embryo. This chapter describes the methods for identifying cellular senescence in the embryo, detailing protocols for the detection of SAβ-gal activity in both sections and at the whole mount level, and immunohistochemistry protocols for the detection of additional biomarkers of senescence.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hayflick L, Moorhead PS (1961) The serial cultivation of human diploid cell strains. Exp Cell Res 25:585–621
Serrano M et al (1997) Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88(5):593–602
Kuilman T et al (2010) The essence of senescence. Genes Dev 24(22):2463–2479
Schmitt CA et al (2002) A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy. Cell 109(3):335–346
Narita M et al (2003) Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence. Cell 113(6):703–716
Zhang R et al (2005) Formation of MacroH2A-containing senescence-associated heterochromatin foci and senescence driven by ASF1a and HIRA. Dev Cell 8(1):19–30
Acosta JC et al (2008) Chemokine signaling via the CXCR2 receptor reinforces senescence. Cell 133(6):1006–1018
Coppe JP et al (2010) The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu Rev Pathol 5:99–118
Kuilman T et al (2008) Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network. Cell 133(6):1019–1031
Braig M et al (2005) Oncogene-induced senescence as an initial barrier in lymphoma development. Nature 436(7051):660–665
Collado M et al (2005) Tumour biology: senescence in premalignant tumours. Nature 436(7051):642
Chen Z et al (2005) Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature 436(7051):725–730
Michaloglou C et al (2005) BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 436(7051):720–724
Baker DJ et al (2011) Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 479(7372):232–236
Campisi J (2013) Aging, cellular senescence, and cancer. Annu Rev Physiol 75:685–705
Storer M et al (2013) Senescence is a developmental mechanism that contributes to embryonic growth and patterning. Cell 155(5):1119–1130
Munoz-Espin D et al (2013) Programmed cell senescence during mammalian embryonic development. Cell 155(5):1104–1118
Krizhanovsky V et al (2008) Senescence of activated stellate cells limits liver fibrosis. Cell 134(4):657–667
Demaria M et al (2014) An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA. Dev Cell 31(6):722–733
Jun JI, Lau LF (2010) The matricellular protein CCN1 induces fibroblast senescence and restricts fibrosis in cutaneous wound healing. Nat Cell Biol 12(7):676–685
Dimri GP et al (1995) A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 92(20):9363–9367
Kurz DJ et al (2000) Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells. J Cell Sci 113(Pt 20):3613–3622
Lee BY et al (2006) Senescence-associated beta-galactosidase is lysosomal beta-galactosidase. Aging Cell 5(2):187–195
Debacq-Chainiaux F et al (2009) Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo. Nat Protoc 4(12):1798–1806
Baker DJ et al (2008) Opposing roles for p16Ink4a and p19Arf in senescence and ageing caused by BubR1 insufficiency. Nat Cell Biol 10(7):825–836
Keyes WM et al (2005) p63 deficiency activates a program of cellular senescence and leads to accelerated aging. Genes Dev 19(17):1986–1999
Brady CA et al (2011) Distinct p53 transcriptional programs dictate acute DNA-damage responses and tumor suppression. Cell 145(4):571–583
Huang T, Rivera-Perez JA (2014) Senescence-associated beta-galactosidase activity marks the visceral endoderm of mouse embryos but is not indicative of senescence. Genesis 52(4):300–308
Acknowledgments
This work was funded by Grants SAF2010-18829 and SAF2013-49082-P to W.M.K. from the Spanish Ministry for Economy and Competitiveness, the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) from the Generalitat de Catalunya, and CRG core funding.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Storer, M., Keyes, W.M. (2017). Detection of Senescence Markers During Mammalian Embryonic Development. In: Nikiforov, M. (eds) Oncogene-Induced Senescence. Methods in Molecular Biology, vol 1534. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6670-7_19
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6670-7_19
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6668-4
Online ISBN: 978-1-4939-6670-7
eBook Packages: Springer Protocols