Name: induction and validation of cellular senescence in primary human cells
Uploaded: 2018-06-20T14:30:00
Description: Watch this Scientific Journal Video about Induction and Validation of Cellular Senescence in Primary Human Cells at JoVE.com

Vi takker medlemmer av The lab for fruktbart diskusjoner og Thijmen van Vliet for deling av data og protokollen på UV-indusert senescence.

1. generelle forberedelser
<ol><li>Forberede D10 medium. Supplere DMEM medium-Glutamax med 10% FBS og 1% penicillin/streptomycin (siste konsentrasjon: 100 U/mL).</li>
	<li>Forberede sterilt PBS. Oppløse tabletter i vann etter produsentens instruksjoner. Sterilisere av autoclave.</li>
	<li>Forberede 1 x trypsin. Fortynne 5 mL av Trypsin-Versene EDTA/10 x 1:10 i 45 mL steril PBS. Merk: Gjennom protokollen, vi bruker celle kultur forhold som er nærmere den fysiologiske forhold for primære fibroblaster. Dette ...

<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. 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Protokollene forklart her var optimalisert for menneskelig primære fibroblaster, spesielt BJ og WI-38 celler. Protokollene for replicative senescence, ioniserende stråling og doksorubicin, har blitt aktivert for andre typer fibroblaster (HCA2 og IMR90) og i andre celletyper (nemlig neonatal melanocytter og keratinocytter eller iPSC-avledet cardiomyocytes) i vårt laboratorium. Imidlertid kan tilpasninger for flere celletyper optimaliseres ved å justere noen detaljer som antall seeded celler, metoder og kjemikalier som...

I 1961 rapportert Hayflick og Moorhead at primære fibroblaster i kultur miste sitt proliferativ potensial etter påfølgende avsnitt1. Denne prosessen er forårsaket av den sekvensielle forkortelsen av telomeres etter hver celledeling. Når telomeres når en kritisk kort lengde, gjenkjennes de av DNA-skade svar (DDR) som aktiverer en irreversibel arrest spredning-også definert som replicative senescence. Replicative senescence er en av mange stimuli som er kjent for å indusere en tilstand av permanente celle syklus arrestasjon som gjengir cellene ufølsomt både mitogens og apoptotisk signaler2,3. Senescence programmet er vanligvis preget av tilleggsfunksjoner inkludert høy lysosomale aktivitet, mitokondrie dysfunksjon, kjernefysiske endringer, chromatin rearrangements, endoplasmatiske retikulum stress, DNA skade og en senescence-assosiert sekretoriske fenotypen (SASP)3,4. Senescent celler har flere funksjoner i kroppen: utvikling, sår helbredelse og tumor undertrykkelse2. Like, de er kjent for å spille en viktig rolle i aldring og paradoksalt nok i svulst progresjon5. Negativ, og delvis motstridende, effekten av senescence er ofte knyttet til SASP6.
Nylig ble det vist at eliminering av senescent celler fra mus fører livslengden utvidelse og eliminering av mange av aldring funksjoner7,8,9,10,11, 12. er utviklet flere medikamenter på samme måte, å enten fjerne senescent celler (senolytics) eller å målrette de SASP13,14. Anti-aging terapeutiske potensialet har nylig fått mer oppmerksomhet til feltet.
Studiet av mekanismer knyttet til mobilnettet senescence og visninger for farmakologiske intervensjoner avhengig tungt av ex vivo modeller, spesielt på menneskelig primære fibroblaster. Mens det er noen fellestrekk aktivert ved ulike senescence indusere, er en stor variasjon i senescence fenotypen observert og avhengig av forskjellige faktorer, inkludert celle type, stimulans og tid punkt3,15, 16,17. Det er viktig å vurdere heterogenitet for å studere og målretting senescent celler. Derfor mål denne protokollen å gi en rekke metoder for å indusere senescence i primære fibroblaster ved hjelp av ulike behandlinger. Som det vil bli forklart, kan metodene lett tilpasses andre celletyper.
Bortsett fra replicative senescence, beskriver vi fem andre senescence-inducing behandlinger: ioniserende stråling, ultrafiolett (UV) stråling, doksorubicin, oksidativt stress og epigenetic endringer (nemlig markedsføringen av histone acetylation eller DNA demethylation) . Både ioniserende stråling og UV-stråling direkte DNA skade og på riktig dose, utløse senescence18,19. Doksorubicin fører også til senescence hovedsakelig gjennom DNA skade av intercalating i DNA og forstyrre topoisomerase II funksjon og dermed stoppe DNA reparasjon mekanismer20. Uttrykket av gener som er avgjørende for senescence er vanligvis styrt av histone acetylation og DNA metylering. Som en konsekvens, utløse histone deacetylase hemmere (f.eks, natrium butyrate og SAHA) og DNA demethylating (f.eks 5-aza) agenter senescence i andre normale celler21,22.
Til slutt, fire av de vanligste markørene knyttet til senescent celler vil bli forklart: aktiviteten til den senescence tilknyttet-β-galactosidase (SA-β-gal), rate av DNA-syntese av EdU innlemmelse analysen, overuttrykte celle syklus regulatorer og cyclin-avhengige kinase hemmere p16 og p21, og overuttrykte og sekresjon av medlemmene av 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&#160;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&#947;-ray machine</td>
		</tr>
		<tr>
			<td>UV radiation chamber</td>
			<td></td>
			<td></td>
			<td>Opsytec, Dr. G&#246;bel BS-02</td>
		</tr>
		<tr>
			<td>Doxorubicin dihydrochloride&#160;</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&#8217;-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&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>B5887</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal (5-Bromo-4-chloro-3-indolyl-&#946;-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&#160;</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&#8217;-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)SO4.5H2O)</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&#160;</td>
			<td>Roche</td>
			<td>4729749001</td>
			<td></td>
		</tr>
		<tr>
			<td>Lightcycler 480 sealing foil&#160;</td>
			<td>Roche</td>
			<td>4729757001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sensifast Probe Lo-ROX kit&#160;</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&#38;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 &#39;M&#39; laboratory film</td>
			<td>Bemis&#160;</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

Mobilnettet senescence er en tilstand av permanente celle syklus arrestasjon aktivert svar på ulike skadelige stimuli. Aktivering av mobilnettet senescence er et kjennetegn på ulike patofysiologiske forhold inkludert svulst undertrykkelse, vev remodeling og aldring. Indusere av mobilnettet senescence i vivo kjennetegnes fortsatt dårlig. En rekke stimuli kan imidlertid brukes til å fremme mobilnettet senescence ex vivo. Blant dem er de vanligste senescence-indusere replicative utmattelse, ioniserende og ikke-ioniserende stråling, gentoksisk narkotika, oksidativt stress, demethylating og acetylerende agenter. Her vil vi gi detaljerte instruksjoner om hvordan du bruker disse stimuli for å indusere fibroblaster til senescence. Denne protokollen kan lett tilpasses for ulike typer primære celler og linjer, inkludert kreftceller. Vi har også beskriver forskjellige metoder for validering av senescence induksjon. Spesielt fokusere vi på å måle aktiviteten til lysosomale enzymet Senescence-assosiert β-galactosidase (SA-β-gal,) frekvensen av DNA-syntese med 5-ethynyl-2-deoxyuridine (EdU) inkorporering analysen, nivåer av uttrykket av cellen syklus hemmere p16 og p21, og uttrykket og sekresjon av medlemmene av Senescence-Associated sekretoriske fenotypen (SASP). Endelig vi gi eksempel resultater og diskutere videre programmer av disse protokollene.

Anriking av SA-β-gal farging i senescent fibroblaster
Β-galactosidase (β-gal) er en lysosomale enzym som er uttrykt i alle celler og som har en optimal pH 4.025,26. Men under senescence, lysosomer øke i størrelse, og følgelig senescent celler akkumulere β-gal. De økte mengder Dette enzymet gjør det mulig å oppdage sin virksomhet på en suboptimal pH 6.0<sup class=...

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

Induksjon og validering av mobilnettet Senescence i primære menneskeceller

Her diskuterer vi en rekke protokoller for induksjon og validering av mobilnettet senescence i kulturperler celler. Vi fokuserer på ulike senescence-inducing stimuli og beskrive kvantifisering av vanlige senescence-assosiert markører. Vi tilbyr tekniske detaljer med fibroblaster som modell, men protokollene kan tilpasses ulike mobilnettet modeller.

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

This method can help answer key questions in the aging and cancer field such as how to perform common techniques to induce cellular senescence and how to evaluate the senescence phenotype. The main advantages of this technique are that it is easy to follow, highly reproducible, does not require any special or expensive equipment, and it results in the induction of senescence in at least 50%of the cellular population. Demonstrating this procedure will be Alejandra Hernandez-Segura, a graduate student from our lab.To begin, seed seven times 10 to the fifth viable primary fibroblasts with a low Population Doubling or PD in a T75 flask containing 10 milliliters of D10. Incubate the cells in a cell culture incubator at 37 degrees Celsius with 5%carbon dioxide and 5%oxygen overnight. Dilute 11 microliters of the 1, 000X doxorubicin stock solution in 11 milliliters of D10 to a final concentration of 250 nanomolar.Then aspirate the medium from the cells and replace it with 10 milliliters of D10 plus doxorubicin. Incubate the cells in a cell culture incubator at 37 degrees Celsius with 5%carbon dioxide and 5%oxygen for exactly 24 hours. Then aspirate the medium and use 10 milliliters of D10 to carefully wash the cells once.Incubate the cells in 10 milliliters of D10 for another six days replacing the medium regularly approximately every three days. To carry out beta-galactosidase staining, for each sample seed one times 10 to the fourth cells in at least one well of a 24-well plate containing 500 microliters of D10 so that the cells are sparse. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide and 5%oxygen overnight.The following day, use 500 microliters of PBS to wash the cells two times. Then add 500 microliters per well of 2%formaldehyde plus 0.2%glutaraldehyde in PBS and incubate the cells at room temperature for three to five minutes to fix them. Then use 500 microliters of PBS to wash the cells twice.Next, after preparing fresh staining solution, add 500 microliters per well and use parafilm to seal the plate to avoid evaporation which can cause crystals to form. Incubate the cells in the dark at 37 degrees Celsius in a dry incubator without carbon dioxide for 12 to 16 hours. Then use 500 microliters of PBS to wash the cells twice.Check the cells under a normal light microscope to assess the results where positive cells will appear with blue paranuclear staining. To facilitate visualization and quantification of individual cells, use DAPI as a counterstain. Then using a fluorescent microscope, determine the percentage of positive cells versus total number of cells taking multiple pictures of the same sample to evaluate at least 100 single cells.For each sample or condition, place one coverslip per well into a 24-well plate. Seed one times 10 to the fourth cells with 500 microliters of D10 per well so that the cells are sparse. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide and 5%oxygen overnight.Prepare enough of a 20 micromolar EdU solution and D10 so that each sample receives 250 microliters. Remove half of the medium from each well being treated and replace it with EdU D10 solution. The final EdU concentration is 10 micromolar.Incubate the samples at 37 degrees Celsius with 5%carbon dioxide and 5%oxygen for 18 to 24 hours. Use the same incubation time for control and senescent cells. After the incubation, use 500 microliters of PBS to wash the cells twice.Then add 500 microliters of 4%formaldehyde in PBS and incubate the cells for 10 minutes to fix them. Following fixation, add 500 microliters of 100 millimolar Tris pH 7.6 to the samples and incubate them for five minutes. Next, add 500 microliters of 0.1%Triton X-100 in PBS to the cells and permeabilize them for 10 minutes.Then use PBS to wash the samples three times. Prepare 50 microliters of label mix for each coverslip, adding the components in the following order, 44.45 microliters of PBS, 0.5 microliters of copper(II)sulfate, 0.05 microliters of sulfo-cy3-azide, and five microliters of sodium ascorbate. Pipette 50 microliters of the label mix on a piece of parafilm.Then using tweezers and a needle, lift the coverslip and rest it cell side down on top of the label mix. Ensure that there are no bubbles and that the whole surface of the coverslip is touching the label mix. Incubate the sample in the dark for 30 minutes.Put the cells back in the wells of the 24-well plate and use PBS to wash them three times. Then use mounting medium with DAPI to mount the samples and let them dry overnight. To begin, seed seven times 10 to the fifth viable primary fibroblasts with a low Population Doubling or.Image at least 100 cells per condition. Finally, quantify the percentage of cells that incorporated EdU by using the following formula. Carry out gene and protein expression studies according to the text protocol.Shown here are representative images of SA-beta-gal staining in proliferating versus senescent primary fibroblasts. Senescent cells look enlarged within a regular cell body. As mentioned, it can be hard to distinguish individual cells.Therefore, co-staining with DAPI facilitates visualization and cell counting. In this experiment, suflo-cy3-azide was used to perform copper catalyzed azide-alkyne cycloaddition to population. If the coupling of the azide to the alkyne has taken place, cells will display fluorescence under a cy3 filter distinguishing proliferating from non-proliferating cells.Once mastered, this technique can be done in 36 hours if performed properly. While attempting this procedure, it is important to remember to be as quick as possible while handling cells outside the incubator as oscillations in the oxygen concentration might affect their results. Following this procedure, other methods like cytokine array or Western blot can be performed in order to answer additional questions like the levels of secreted proteins or understanding specific signaling pathways.After its development, this technique paved the way for researchers in the aging and cancer field to explore how senescent cells are involved in physiological and pathological conditions in mammals. After watching this video, you should have a good understanding on how to reproducibly induce senescence in primary cells and how to assess their senescence status.

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