We thank members of the Demaria lab for fruitful discussions, and Thijmen van Vliet for sharing data and protocol on the UV-induced senescence.

<ol>
	<li>Hayflick, L., Moorhead, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+serial+cultivation+of+human+diploid+cell+strains.">The serial cultivation of human diploid cell strains.</a> Experimental Cell Research. 25, 585-621 (1961).</li><li>Mu&#241;oz-Esp&#237;n, D., Serrano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+senescence:+from+physiology+to+pathology.+Nature+reviews.">Cellular senescence: from physiology to pathology. Nature reviews.</a> Molecular cell biology. 15, 482-496 (2014).</li><li>Sharpless, N. E., Sherr, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forging+a+signature+of+in+vivo+senescence.">Forging a signature of in vivo senescence.</a> Nature Reviews Cancer. 15 (7), 397-408 (2015).</li><li>Correia-Melo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+are+required+for+pro-ageing+features+of+the+senescent+phenotype.">Mitochondria are required for pro-ageing features of the senescent phenotype.</a> The EMBO Journal. 10, e201592862 (2016).</li><li>Loaiza, N., Demaria, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+senescence+and+tumor+promotion:+Is+aging+the+key?.">Cellular senescence and tumor promotion: Is aging the key?.</a> Biochimica et Biophysica Acta (BBA) - Reviews on Cancer. , (2016).</li><li>Coppe, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescence-associated+secretory+phenotypes+reveal+cell-nonautonomous+functions+of+oncogenic+RAS+and+the+p53+tumor+suppressor.">Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor.</a> PLoS Biology. 6 (12), 2853-2868 (2008).</li><li>Baker, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+occurring+p16(Ink4a)-positive+cells+shorten+healthy+lifespan.">Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan.</a> Nature. 530 (7589), 184-189 (2016).</li><li>Xu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+senescent+cells+enhances+adipogenesis+and+metabolic+function+in+old+age.">Targeting senescent cells enhances adipogenesis and metabolic function in old age.</a> Elife. 4, e12997 (2015).</li><li>Baker, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clearance+of+p16Ink4a-positive+senescent+cells+delays+ageing-associated+disorders.">Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders.</a> Nature. 479 (7372), 232-236 (2011).</li><li>Jeon, O. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+clearance+of+senescent+cells+attenuates+the+development+of+post-traumatic+osteoarthritis+and+creates+a+pro-regenerative+environment.">Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment.</a> Nature Medicine. 23 (6), 775-781 (2017).</li><li>Demaria, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Senescence+Promotes+Adverse+Effects+of+Chemotherapy+and+Cancer+Relapse.">Cellular Senescence Promotes Adverse Effects of Chemotherapy and Cancer Relapse.</a> Cancer Discovery. 7 (2), 165-176 (2017).</li><li>Chang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clearance+of+senescent+cells+by+ABT263+rejuvenates+aged+hematopoietic+stem+cells+in+mice.">Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice.</a> Nature Medicine. 22 (1), 78-83 (2016).</li><li>Soto-Gamez, A., Demaria, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+interventions+for+aging:+the+case+of+cellular+senescence.">Therapeutic interventions for aging: the case of cellular senescence.</a> Drug Discov Today. 22 (5), 786-795 (2017).</li><li>Childs, B. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescent+cells:+an+emerging+target+for+diseases+of+ageing.">Senescent cells: an emerging target for diseases of ageing.</a> Nature Reviews Drug Discovery. 16 (10), 718-735 (2017).</li><li>Marthandan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+genes+and+pathways+in+primary+human+fibroblast+strains+undergoing+replicative+and+radiation+induced+senescence.">Conserved genes and pathways in primary human fibroblast strains undergoing replicative and radiation induced senescence.</a> Biological Research. 49, 34 (2016).</li><li>Marthandan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+Senescence+Associated+Genes+and+Pathways+in+Primary+Human+Fibroblasts+Detected+by+RNA-Seq.">Conserved Senescence Associated Genes and Pathways in Primary Human Fibroblasts Detected by RNA-Seq.</a> PLoS One. 11 (5), e0154531 (2016).</li><li>Hernandez-Segura, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unmasking+Transcriptional+Heterogeneity+in+Senescent+Cells.">Unmasking Transcriptional Heterogeneity in Senescent Cells.</a> Current Biology. 27 (17), 2652-2660 (2017).</li><li>Le, O. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionizing+radiation-induced+long-term+expression+of+senescence+markers+in+mice+is+independent+of+p53+and+immune+status.">Ionizing radiation-induced long-term expression of senescence markers in mice is independent of p53 and immune status.</a> Aging Cell. 9 (3), 398-409 (2010).</li><li>Hall, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C/EBPalpha+regulates+CRL4(Cdt2)-mediated+degradation+of+p21+in+response+to+UVB-induced+DNA+damage+to+control+the+G1/S+checkpoint.">C/EBPalpha regulates CRL4(Cdt2)-mediated degradation of p21 in response to UVB-induced DNA damage to control the G1/S checkpoint.</a> Cell Cycle. 13 (22), 3602-3610 (2014).</li><li>Nitiss, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+DNA+topoisomerase+II+in+cancer+chemotherapy.">Targeting DNA topoisomerase II in cancer chemotherapy.</a> Nature Reviews Cancer. 9 (5), 338-350 (2009).</li><li>Pazolli, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+remodeling+underlies+the+senescence-+associated+secretory+phenotype+of+tumor+stromal+fibroblasts+that+supports+cancer+progression.">Chromatin remodeling underlies the senescence- associated secretory phenotype of tumor stromal fibroblasts that supports cancer progression.</a> Cancer Research. 72, 2251-2261 (2012).</li><li>Venturelli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+induction+of+apoptosis+and+senescence+by+the+DNA+methyltransferase+inhibitors+5-azacytidine+and+5-aza-2'-deoxycytidine+in+solid+tumor+cells.">Differential induction of apoptosis and senescence by the DNA methyltransferase inhibitors 5-azacytidine and 5-aza-2'-deoxycytidine in solid tumor cells.</a> Molecular Cancer Therapeutics. 12, 2226-2236 (2013).</li><li>Tennant, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+Trypan+Blue+Technique+for+Determination+of+Cell+Viability.">Evaluation of the Trypan Blue Technique for Determination of Cell Viability.</a> Transplantation. 2, 685-694 (1964).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Lee, B. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescence-associated+&#946;-galactosidase+is+lysosomal+&#946;-galactosidase.">Senescence-associated &#946;-galactosidase is lysosomal &#946;-galactosidase.</a> Aging Cell. 5, 187-195 (2006).</li><li>Kopp, H. G., Hooper, A. T., Shmelkov, S. V., Rafii, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-galactosidase+staining+on+bone+marrow.+The+osteoclast+pitfall.">Beta-galactosidase staining on bone marrow. The osteoclast pitfall.</a> Histology and Histopathology. 22 (9), 971-976 (2007).</li><li>Dimri, G. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biomarker+that+identifies+senescent+human+cells+in+culture+and+in+aging+skin+in+vivo.">A biomarker that identifies senescent human cells in culture and in aging skin in vivo.</a> Proceedings of the National Academy of Sciences. 92 (20), 9363-9367 (1995).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proceedings of the National Academy of Sciences. 105 (7), 2415-2420 (2008).</li><li>Sherr, C. J., McCormick, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RB+and+p53+pathways+in+cancer.">The RB and p53 pathways in cancer.</a> Cancer Cell. 2 (2), 103-112 (2002).</li><li>Bunz, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+p53+and+p21+to+sustain+G2+arrest+after+DNA+damage.">Requirement for p53 and p21 to sustain G2 arrest after DNA damage.</a> Science. 282 (5393), 1497-1501 (1998).</li><li>Severino, J., Allen, R. G., Balin, S., Balin, A., Cristofalo, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+beta-galactosidase+staining+a+marker+of+senescence+in+vitro+and+in+vivo.">Is beta-galactosidase staining a marker of senescence in vitro and in vivo.</a> Experimental Cell Research. 257 (1), 162-171 (2000).</li><li>Stolzing, A., Coleman, N., Scutt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose-induced+replicative+senescence+in+mesenchymal+stem+cells.">Glucose-induced replicative senescence in mesenchymal stem cells.</a> Rejuvenation Research. 9 (1), 31-35 (2006).</li><li>Blazer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+glucose-induced+replicative+senescence:+point+of+no+return+and+effect+of+telomerase.">High glucose-induced replicative senescence: point of no return and effect of telomerase.</a> Biochemical and Biophysical Research Communications. 296 (1), 93-101 (2002).</li><li>Wiley, C. D., Campisi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Ancient+Pathways+to+Aging+Cells-Connecting+Metabolism+and+Cellular+Senescence.">From Ancient Pathways to Aging Cells-Connecting Metabolism and Cellular Senescence.</a> Cell Metabolism. 23 (6), 1013-1021 (2016).</li><li>Kumar, R., Gont, A., Perkins, T. J., Hanson, J. E. L., Lorimer, I. A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+senescence+in+primary+glioblastoma+cells+by+serum+and+TGFbeta.">Induction of senescence in primary glioblastoma cells by serum and TGFbeta.</a> Scientific Reports. 7 (1), 2156 (2017).</li><li>Hypoxia Blagosklonny, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MTOR+and+autophagy:+converging+on+senescence+or+quiescence.">MTOR and autophagy: converging on senescence or quiescence.</a> Autophagy. 9 (2), 260-262 (2013).</li><li>Meuter, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+of+cellular+senescence+are+elevated+in+murine+blastocysts+cultured+in+vitro:+molecular+consequences+of+culture+in+atmospheric+oxygen.">Markers of cellular senescence are elevated in murine blastocysts cultured in vitro: molecular consequences of culture in atmospheric oxygen.</a> Journal of Assisted Reproduction and Genetics. 31 (10), 1259-1267 (2014).</li><li>Coppe, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human-like+senescence-associated+secretory+phenotype+is+conserved+in+mouse+cells+dependent+on+physiological+oxygen.">A human-like senescence-associated secretory phenotype is conserved in mouse cells dependent on physiological oxygen.</a> PLoS One. 5 (2), e9188 (2010).</li><li>van Deursen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+senescent+cells+in+ageing.">The role of senescent cells in ageing.</a> Nature. 509 (7501), 439-446 (2014).</li><li>Kim, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implications+of+time-series+gene+expression+profiles+of+replicative+senescence.">Implications of time-series gene expression profiles of replicative senescence.</a> Aging Cell. 12, 622-634 (2013).</li></ol>

The protocols explained here were optimized for human primary fibroblasts, particularly BJ and WI-38 cells. The protocols for replicative senescence, ionizing radiation and doxorubicin, have been successfully applied to other types of fibroblasts (HCA2 and IMR90) and in other cell types (namely neonatal melanocytes and keratinocytes or iPSC-derived cardiomyocytes) in our laboratory. However, adaptations for additional cell types can be optimized by adjusting some details such as the number of seeded cells, the methods an...

In 1961, Hayflick and Moorhead reported that primary fibroblasts in culture lose their proliferative potential after successive passages1. This process is caused by the sequential shortening of telomeres after each cell division. When telomeres reach a critically short length, they are recognized by the DNA-damage response (DDR) that activates an irreversible arrest of proliferation &#8212; also defined as replicative senescence. Replicative senescence is currently one of the many stimuli that are known to induce a state of permanent cell cycle arrest that renders cells insensitive both to mitogens and to apoptotic signals2,3. The senescence program is normally characterized by additional features including high lysosomal activity, mitochondrial dysfunction, nuclear changes, chromatin rearrangements, endoplasmic reticulum stress, DNA damage and a senescence-associated secretory phenotype (SASP)3,4. Senescent cells have multiple functions in the body: development, wound healing and tumor suppression2. Equally, they are known to play an important role in aging and, paradoxically, in tumor progression5. The negative, and partially contradictory, effects of senescence are often attributed to the SASP6.
Recently, it was shown that elimination of senescent cells from mice leads to lifespan extension and to elimination of many of the aging features7,8,9,10,11,12. In the same way, multiple drugs have been developed to either eliminate senescent cells (senolytics) or to target the SASP13,14. The anti-aging therapeutic potential has recently attracted more attention to the field.
The study of mechanisms associated to cellular senescence and the screenings for pharmacological interventions heavily rely on ex vivo models, particularly on human primary fibroblasts. While there are some common features activated by diverse senescence inducers, a large variability in the senescence phenotype is observed and dependent on various factors including cell type, stimulus and time point3,15,16,17. It is imperative to consider the heterogeneity for studying and targeting senescent cells. Therefore, this protocol aims to provide a series of methods used to induce senescence in primary fibroblasts by using different treatments. As it will be explained, the methods can easily be adapted to other cell types.
Apart from replicative senescence, we describe five other senescence-inducing treatments: ionizing radiation, ultraviolet (UV) radiation, doxorubicin, oxidative stress and epigenetic changes (namely promotion of histone acetylation or DNA demethylation). Both, ionizing radiation and UV-radiation cause direct DNA damage and, at the appropriate dose, trigger senescence18,19. Doxorubicin also causes senescence mainly through DNA damage by intercalating into the DNA and disrupting topoisomerase II function and thus halting DNA repair mechanisms20. The expression of genes essential for senescence is normally controlled by histone acetylation and DNA methylation. As a consequence, histone deacetylase inhibitors (e.g., sodium butyrate and SAHA) and DNA demethylating (e.g., 5-aza) agents trigger senescence in otherwise normal cells21,22.
Finally, four of the most common markers associated to senescent cells will be explained: activity of the senescence associated-&#946;-galactosidase (SA-&#946;-gal), rate of DNA synthesis by EdU incorporation assay, overexpression of the cell cycle regulators and cyclin-dependent kinase inhibitors p16 and p21, and overexpression and secretion of members of the SASP.

<table><tbody><tr><td>DMEM Media - GlutaMAX</td><td>Gibco</td><td>31966-047</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SV30160.03</td><td></td></tr><tr><td>Penicillin-Streptomycin (P/S; 10,000 U/ml)</td><td>Lonza</td><td>DE17-602E</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>SC-202581</td><td></td></tr><tr><td>Nuclease-Free Water (not DEPC-Treated)</td><td>Ambion</td><td>AM9937</td><td></td></tr><tr><td>T75 flask</td><td>Sarstedt</td><td>833911002</td><td></td></tr><tr><td>Trypsin/EDTA Solution</td><td>Lonza</td><td>CC-5012</td><td></td></tr><tr><td>PBS tablets</td><td>Gibco</td><td>18912-014</td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>Sigma-Aldrich</td><td>T9661-1000EA</td><td></td></tr><tr><td>Corning&amp;nbsp;15 mL centrifuge tubes</td><td>Sigma-Aldrich</td><td>CLS430791</td><td></td></tr><tr><td>6-well plate</td><td>Sarstedt</td><td>83.3920</td><td></td></tr><tr><td>24-well plate</td><td>Sarstedt</td><td>83.3922</td><td></td></tr><tr><td>13mm round coverslips</td><td>Sarstedt</td><td>83.1840.002</td><td></td></tr><tr><td>Steriflip</td><td>Merck Chemicals</td><td>SCGP00525</td><td></td></tr><tr><td>Cesium137-source</td><td></td><td></td><td>IBL 637 Cesium-137&amp;gamma;-ray machine</td></tr><tr><td>UV radiation chamber</td><td></td><td></td><td>Opsytec, Dr. G&amp;ouml;bel BS-02</td></tr><tr><td>Doxorubicin dihydrochloride&amp;nbsp;</td><td>BioAustralis Fine Chemicals</td><td>BIA-D1202-1</td><td></td></tr><tr><td>Hydrogen peroxide solution</td><td>Sigma-Aldrich</td><td>7722-84-1</td><td></td></tr><tr><td>5-aza-2&amp;rsquo;-deoxycytidine</td><td>Sigma-Aldrich</td><td>A3656</td><td></td></tr><tr><td>SAHA</td><td>Sigma-Aldrich</td><td>SML0061</td><td></td></tr><tr><td>Sodium Butyrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>B5887</td><td></td></tr><tr><td>X-gal (5-Bromo-4-chloro-3-indolyl-&amp;beta;-D-galactopyranoside)</td><td>Fisher Scientific</td><td>7240-90-6</td><td></td></tr><tr><td>Citric acid monohydrate</td><td>Sigma-Aldrich</td><td>5949-29-1</td><td></td></tr><tr><td>Sodium dibasic phosphate</td><td>Acros organics</td><td>7782-85-6</td><td></td></tr><tr><td>Potassium ferrocyanide&amp;nbsp;</td><td>Fisher Scientific</td><td>14459-95-1</td><td></td></tr><tr><td>Potassium ferricyanide</td><td>Fisher Scientific</td><td>13746-66-2</td><td></td></tr><tr><td>Sodium Chloride</td><td>Merck Millipore</td><td>7647-14-5</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Fisher Chemicals</td><td>7791-18-6</td><td></td></tr><tr><td>25% glutaraldehyde</td><td>Fisher Scientific</td><td>111-30-8,7732-18-5</td><td></td></tr><tr><td>16% formaldehyde (w/v)</td><td>Thermo-Fisher Scientific</td><td>28908</td><td></td></tr><tr><td>EdU (5-ethynyl-2&amp;rsquo;-deoxyuridine)</td><td>Lumiprobe</td><td>10540</td><td></td></tr><tr><td>Sulfo-Cyanine3 azide (Sulfo-Cy3-Azide)</td><td>Lumiprobe</td><td>D1330</td><td></td></tr><tr><td>Sodium ascorbate</td><td>Sigma-Aldrich</td><td>A4034</td><td></td></tr><tr><td>Copper(II) sulfate pentahydrate (Cu(II)SO&lt;sub&gt;4&lt;/sub&gt;.5H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>209198</td><td></td></tr><tr><td>Triton X-100</td><td>Acros organics</td><td>215682500</td><td></td></tr><tr><td>TRIS base</td><td>Roche</td><td>11814273001</td><td></td></tr><tr><td>LightCycler 480 Multiwell Plate 384, white&amp;nbsp;</td><td>Roche</td><td>4729749001</td><td></td></tr><tr><td>Lightcycler 480 sealing foil&amp;nbsp;</td><td>Roche</td><td>4729757001</td><td></td></tr><tr><td>Sensifast Probe Lo-ROX kit&amp;nbsp;</td><td>Bioline</td><td>BIO-84020</td><td></td></tr><tr><td>UPL Probe Library</td><td>Sigma-Aldrich</td><td>Various</td><td></td></tr><tr><td>Human IL-6 DuoSet ELISA</td><td>R&amp;amp;D</td><td>D6050</td><td></td></tr><tr><td>Bio-Rad TC20</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Counting slides</td><td>Bio-Rad</td><td>145-0017</td><td></td></tr><tr><td>Dry incubator</td><td>Thermo-Fisher Scientific</td><td>Heratherm</td><td></td></tr><tr><td>Dimethylformamide</td><td>Merck Millipore</td><td>1.10983</td><td></td></tr><tr><td>Parafilm &#039;M&#039; laboratory film</td><td>Bemis&amp;nbsp;</td><td>#PM992</td><td></td></tr><tr><td>Tweezers</td><td></td><td></td><td></td></tr><tr><td>Needles</td><td></td><td></td><td></td></tr></tbody></table>

induction and validation of cellular senescence in primary human cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Enrichment of SA-&#946;-gal staining in senescent fibroblasts
&#914;-galactosidase (&#946;-gal) is a lysosomal enzyme that is expressed in all cells and that has an optimum pH of 4.025,26. However, during senescence, lysosomes increase in size and, consequently, senescent cells accumulate &#946;-gal. The increased amounts of this enzyme make it possible to detect its activi...

Watch this Scientific Journal Video about Induction and Validation of Cellular Senescence in Primary Human Cells at JoVE.com

Induction and Validation of Cellular Senescence in Primary Human Cells

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

induction-and-validation-of-cellular-senescence-in-primary-human-cells

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Cell Proliferation

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16.7K Views.  University of Groningen, University Medical Center Groningen. Hier bespreken we een serie van protocollen voor de inductie en validatie van cellulaire senescentie in gekweekte cellen. Wij richten op verschillende senescentie-inducerende prikkels en de kwantificering van gemeenschappelijke senescentie-geassocieerde markers te beschrijven. Wij bieden technische details met behulp van fibroblasten als een model, maar de protocollen kunnen worden aangepast aan verschillende cellulaire modellen.

Video: Induction and Validation of Cellular Senescence in Primary Human Cells

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field of regenerative medicine. However, despite their benefits, the tumorigenic potential of ESCs and iPSCs has long been a barrier for clinical applications. Interestingly, it has been shown that ESCs produce several soluble factors that can promote tissue regeneration and delay cellular aging, suggesting that ESCs and iPSCs can also be utilized as a cell-free intervention method. Therefore, the method for harvesting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) with minimal contamination of serum components (fetal bovine serum, FBS) and feeder cells (mouse embryonic fibroblasts, MEFs) has been highly demanded. Here, the present study demonstrates an optimized method for the collection of mESC-CM under serum- and feeder-free conditions and for the characterization of mESC-CM using senescence-associated multiple readouts. This protocol will provide a method to collect pure mESC-specific secretory factors without serum and feeder contamination.

This protocol provides a method for the collection of mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) derived from serum (fetal bovine serum, FBS)- and feeder (mouse embryonic fibroblasts, MEFs)-free conditions for a cell-free approach. It may be applicable for the treatment of aging and aging-associated diseases.

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

A Sensitive Method to Quantify Senescent Cancer Cells

Human cells do not indefinitely proliferate. Upon external and/or intrinsic cues, cells might die or enter a stable cell cycle arrest called senescence. Several cellular mechanisms, such as telomere shortening and abnormal expression of mitogenic oncogenes, have been shown to cause senescence. Senescence is not restricted to normal cells; cancer cells have also been reported to senesce.
	Chemotherapeutical drugs have been shown to induce senescence in cancer cells. However, it remains controversial whether senescence prevents or promotes tumorigenesis. As it might eventually be patient-specific, a rapid and sensitive method to assess senescence in cancer cell will soon be required.
	To this end, the standard &beta;-galactosidase assay, the currently used method, presents major drawbacks: it is time consuming and not sensitive. We propose here a flow cytometry-based assay to study senescence on live cells. This assay offers the advantage of being rapid, sensitive, and can be coupled to the immunolabeling of various cellular markers.

Whether senescence prevents or promotes tumorigenesis remains controversial. Since chemotherapeutical drugs can induce cancer cells to senesce, studying senescence is essential for proposing new therapies. However, the standard and broadly used &beta;-galactosidase assay presents major drawbacks. We propose here a rapid and sensitive flow cytometry-based assay to quantify senescence.

Human cells do not indefinitely proliferate. Upon external and/or intrinsic  cues, cells might die or enter a stable cell cycle arrest called senescence. ...

Medicine

A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. Recent studies have extended the role of cellular senescence to various physiological processes, including development, wound healing, immune surveillance, and age-related tissue dysfunction. Although cell cycle arrest is a critical hallmark of cellular senescence, an increased intracellular reactive oxygen species (ROS) production has also been demonstrated to play an important role in the induction of cellular senescence. In addition, recent studies revealed that senescent cells exhibit potent paracrine activities on neighboring cells and tissues through a senescence-associated secretory phenotype (SASP). The sharp increase in interest regarding therapeutic strategies against cellular senescence emphasizes the need for a precise understanding of senescence mechanisms, including intracellular ROS and the SASP. Here, we describe protocols for quantitatively assessing intracellular ROS levels during H-Ras-induced cellular senescence using ROS-sensitive fluorescent dye and flow cytometry. In addition, we introduce sensitive techniques for the analysis of the induction of mRNA expression and secretion of SASP factors. These protocols can be applied to various cellular senescence models.

Intracellular ROS has been shown to play an important role in the induction of cellular senescence. Here, we describe a sensitive assay for quantifying ROS levels during cellular senescence. We also provide protocols for assessing the senescence-associated secretory phenotype, which reportedly contributes to various age-related dysfunctions.

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

Cancer Research

Far-Red Fluorescent Senescence-Associated &#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge rapidly in tumor cells as a response to damage induced by various cancer treatments. Tumor cell senescence is generally considered undesirable, as senescent cells become resistant to death and block tumor remission while exacerbating tumor malignancy and treatment resistance. Therefore, the identification of senescent tumor cells is of ongoing interest to the cancer research community. Various senescence assays exist, many based on the activity of the well-known senescence marker, senescence-associated beta-galactosidase (SA-&#946;-Gal). 
Typically, the SA-&#946;-Gal assay is performed using a chromogenic substrate (X-Gal) on fixed cells, with the slow and subjective enumeration of "blue" senescent cells by light microscopy. Improved assays using cell-permeant, fluorescent SA-&#946;-Gal substrates, including C12-FDG (green) and DDAO-Galactoside (DDAOG; far-red), have enabled the analysis of living cells and allowed the use of high-throughput fluorescent analysis platforms, including flow cytometers. C12-FDG is a well-documented probe for SA-&#946;-Gal, but its green fluorescent emission overlaps with intrinsic cellular autofluorescence (AF) that arises during senescence due to the accumulation of lipofuscin aggregates. By utilizing the far-red SA-&#946;-Gal probe DDAOG, green cellular autofluorescence can be used as a secondary parameter to confirm senescence, adding reliability to the assay. The remaining fluorescence channels can be used for cell viability staining or optional fluorescent immunolabeling.
Using flow cytometry, we demonstrate the use of DDAOG and lipofuscin autofluorescence as a dual-parameter assay for the identification of senescent tumor cells. Quantitation of the percentage of viable senescent cells is performed. If desired, an optional immunolabeling step may be included to evaluate cell surface antigens of interest. Identified senescent cells can also be flow cytometrically sorted and collected for downstream analysis. Collected senescent cells can be immediately lysed (e.g., for immunoassays or 'omics analysis) or further cultured.

A protocol for fluorescent, flow cytometric quantification of senescent cancer cells induced by chemotherapy drugs in cell culture or in murine tumor models is presented. Optional procedures include co-immunostaining, sample fixation to facilitate large batch or time point analysis, and the enrichment of viable senescent cells by flow cytometric sorting.

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge ...

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as Alzheimer&#39;s and Parkinson&#39;s diseases. Cellular senescence is a state of permanent cell cycle arrest that typically occurs in response to exposure to sub-lethal stresses. However, like other non-dividing cells, senescent cells remain metabolically active and carry out many functions that require unique transcriptional and translational demands and widespread changes in the intracellular and secreted proteomes. Understanding how protein synthesis and decay rates change during senescence can illuminate the underlying mechanisms of cellular senescence and find potential therapeutic avenues for diseases exacerbated by senescent cells. This paper describes a method for proteome-scale evaluation of protein half-lives in non-dividing cells using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) in combination with mass spectrometry. pSILAC involves metabolic labeling of cells with stable heavy isotope-containing versions of amino acids. Coupled with modern mass spectrometry approaches, pSILAC enables the measurement of protein turnover of hundreds or thousands of proteins in complex mixtures. After metabolic labeling, the turnover dynamics of proteins can be determined based on the relative enrichment of heavy isotopes in peptides detected by mass spectrometry. In this protocol, a workflow is described for the generation of senescent fibroblast cultures and similarly arrested quiescent fibroblasts, as well as a simplified, single-time point pSILAC labeling time-course that maximizes coverage of anticipated protein turnover rates. Further, a pipeline is presented for the analysis of pSILAC mass spectrometry data and user-friendly calculation of protein degradation rates using spreadsheets. The application of this protocol can be extended beyond senescent cells to any non-dividing cultured cells such as neurons.

This protocol describes the workflow for metabolic labeling of senescent and non-dividing cells with pulsed SILAC, untargeted mass spectrometry analysis, and a streamlined calculation of protein half-lives.

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as ...

Biochemistry

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

Biology

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Induction and Validation of Cellular Senescence in Primary Human Cells

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