Wir danken Mitglieder des Demaria Labors für fruchtbare Diskussionen und Thijmen van Vliet für den Austausch von Daten und das Protokoll auf der UV-induzierten Seneszenz.

<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. 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Die hier erläuterten Protokolle wurden optimiert für menschliche primäre Fibroblasten, besonders BJ und WI-38 Zellen. Die Protokolle für replikative Seneszenz, ionisierende Strahlung und Doxorubicin, worden erfolgreich angewandt, um andere Arten von Fibroblasten (HCA2 und IMR90) und in andere Zelltypen (nämlich neonatale Melanozyten und Keratinozyten oder iPSC-abgeleitete Herzmuskelzellen) in unserem Labor. Anpassungen für weitere Zelltypen können jedoch durch Anpassung einige Details wie die Anzahl der gesetzten ...

Im Jahr 1961 berichtet Hayflick und Moorhead, dass primäre Fibroblasten in Kultur ihr proliferative Potential nach aufeinanderfolgenden Passagen1zu verlieren. Dieser Prozess wird durch die fortlaufende Verkürzung der Telomere nach jeder Zellteilung verursacht. Wenn die Telomere eine kritisch kurze Länge erreichen, sie sind anerkannt durch die DNA-Schäden-Antwort (DDR), die eine irreversible Verhaftung der Proliferation aktiviert – auch als replikative Seneszenz definiert. Replikative Seneszenz ist derzeit eines der vielen Reize, die bekannt sind, um einen Zustand der permanenten Zellzyklus Festnahme zu induzieren, die Zellen unempfindlich auf Mitogene und Apoptotic Signale2,3 macht. Die Seneszenz-Programm zeichnet sich normalerweise durch zusätzliche Eigenschaften einschließlich lysosomale hochaktiver, mitochondriale Dysfunktion, nukleare Änderungen, Chromatin Umgestaltungen, endoplasmatische Retikulum Stress, DNA-Schäden und Seneszenz-assoziierten sekretorischen Phänotyp (SASP)3,4. Alternde Zellen haben mehrere Funktionen im Körper: Entwicklung, Wunde heilen und Tumor Unterdrückung2. Ebenso sind sie bekannt, eine wichtige Rolle im Altern und paradoxerweise im Tumor Progression5. Die negativen und teilweise widersprüchlichen Auswirkungen der Seneszenz werden oft die SASP6zugeschrieben.
Vor kurzem zeigte sich, dass die Beseitigung der alternde Zellen von Mäusen zur Lebensdauerverlängerung und zur Beseitigung vieler der Alterung Funktionen7,8,9,10,11, führt 12. auf die gleiche Weise wurden mehrere Medikamente entwickelt, alternde Zellen (Senolytics) entweder zu beseitigen oder die SASP13,14Zielen. Das therapeutische Potenzial von Anti-Aging hat vor kurzem mehr Aufmerksamkeit auf das Feld angezogen.
Die Untersuchung der Mechanismen, die zelluläre Seneszenz zugeordnet und die Vorführungen für pharmakologische Interventionen angewiesen auf ex-Vivo -Modelle, vor allem auf menschliche primäre Fibroblasten. Zwar gibt es einige gemeinsame Merkmale, die durch vielfältige Seneszenz Induktoren aktiviert, ist eine große Variabilität in der Seneszenz Phänotyp beobachteten und abhängig von verschiedenen Faktoren ab, einschließlich der Zelle Art, Impuls und Zeit Punkt3,15, 16,17. Es ist zwingend notwendig, um die Heterogenität für Studium und targeting seneszenten Zellen betrachten. Dieses Protokoll soll daher eine Reihe von Methoden zur Seneszenz in primäre Fibroblasten zu induzieren, indem mit verschiedenen Behandlungen zu bieten. Wie es erklärt wird, können die Methoden leicht zu anderen Zelltypen angepasst werden.
Abgesehen von replikative Seneszenz, beschreiben wir fünf Seneszenz-induzierende Behandlungen: ionisierende Strahlung, ultraviolettes (UV) Strahlung, Doxorubicin, oxidativer Stress und epigenetische Veränderungen (nämlich die Förderung von Histon Acetylierung oder DNA Demethylierung) . Ionisierende Strahlung sowohl UV-Bestrahlung direkte DNA-Schäden verursachen, und bei der entsprechenden Dosis auslösen Seneszenz18,19. Doxorubicin verursacht auch Seneszenz hauptsächlich durch DNA-Schäden durch verschuppen in die DNA und Topoisomerase-II-Funktion zu stören und damit stoppen DNA Reparatur Mechanismen20. Die Expression von Genen für Seneszenz wird normalerweise von Histon Acetylierung und DNA-Methylierung gesteuert. Folglich auslösen Histon-Deacetylase-Hemmer (z.B.Natrium-Butyrat und SAHA) und DNA-demethylating (z. B. 5-Aza) Agenten Seneszenz sonst normalen Zellen21,22.
Zu guter letzt vier der am häufigsten verwendeten Marker, die alternde Zellen erläutert werden: Aktivität der Seneszenz verbunden-β-Galaktosidase (SA-β-gal), Rate der DNA-Synthese durch EdU Einbeziehung Assay, Überexpression von den Regulatoren des Zellzyklus und Cyclin-abhängige Kinase-Inhibitoren p16 p21, Überexpression und und Sekretion von Mitgliedern der 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

Zelluläre Seneszenz ist ein Zustand des permanenten Zellzyklus Festnahme in Reaktion auf verschiedene schädliche Reize aktiviert. Aktivierung der zellulären Seneszenz ist ein Markenzeichen von diversen pathophysiologischen Bedingungen auch den Tumor Unterdrückung, Gewebe, Umbau und Alterung. Die Induktoren der zellulären Seneszenz in Vivo zeichnen sich nach wie vor schlecht. Eine Reihe von Reizen kann jedoch verwendet werden, Förderung der zellulären Seneszenz ex Vivo. Darunter sind die häufigsten Seneszenz-Induktoren replikativen Erschöpfung, ionisierender und nichtionisierender Strahlung, genotoxisch Drogen, oxidativen Stress und demethylating und acetylieren Agenten. Hier bieten wir detaillierte Anweisungen zur Verwendung dieser Reize, um Fibroblasten in Seneszenz zu induzieren. Dieses Protokoll kann problemlos für verschiedene Arten von Primärzellen und Zelllinien, einschließlich Krebszellen angepasst werden. Wir beschreiben auch verschiedene Methoden für die Validierung der Seneszenz Induktion. Insbesondere konzentrieren wir uns auf die Messung der Aktivität des lysosomalen Enzyms Seneszenz-assoziierten β-Galaktosidase (SA-β-gal), die Rate der DNA-Synthese mit 5-Ethynyl-2'-Deoxyuridine (EdU) Einbeziehung Assay, die Höhe der Expression des Zellzyklus Inhibitoren p16 p21, und der Ausdruck und Sekretion von Mitgliedern der Senescence-Associated sekretorischen Phänotyp (SASP). Endlich, wir bieten Beispielergebnisse und besprechen weitere Anwendungen dieser Protokolle.

Bereicherung der SA-β-gal-Färbung in seneszenten Fibroblasten
Β-Galaktosidase (β-gal) ist eine lysosomale Enzym, das drückt sich in allen Zellen und hat einen optimalen pH-Wert von 4,025,26. Jedoch während der Seneszenz, Lysosomen an Größe zunehmen und infolgedessen seneszenten Zellen ansammeln β-gal. Die erhöhten Mengen dieses Enzyms machen es möglich, seine Tä...

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

Induktion und Validierung der zellulären Seneszenz in primären humanen Zellen

Hier diskutieren wir eine Reihe von Protokollen für die Induktion und Validierung der zellulären Seneszenz in kultivierten Zellen. Wir konzentrieren uns auf unterschiedlichen Seneszenz-induzierende Stimuli und beschreiben die Quantifizierung der gemeinsamen Seneszenz-assoziierten Marker. Wir bieten technische Details mit Fibroblasten als Modell, aber die Protokolle können verschiedene zelluläre Modelle angepasst werden.

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 Biology

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Unit 1

Cell Proliferation

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16.6K Aufrufe.  University of Groningen, University Medical Center Groningen. Hier diskutieren wir eine Reihe von Protokollen für die Induktion und Validierung der zellulären Seneszenz in kultivierten Zellen. Wir konzentrieren uns auf unterschiedlichen Seneszenz-induzierende Stimuli und beschreiben die Quantifizierung der gemeinsamen Seneszenz-assoziierten Marker. Wir bieten technische Details mit Fibroblasten als Modell, aber die Protokolle können verschiedene zelluläre Modelle angepasst werden.

Serie: 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.

Sammlung von Serum- und Feeder-freie Maus embryonale Zellen konditionierten Medium für eine zellfreie Ansatz Stem

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 ...

Forschung

Entwicklungsbiologie

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.

Bewertung von Verletzungen-Induzierte Seneszenz und In Vivo eine Anpassung in der Skelettmuskulatur

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.

Eine empfindliche Methode zur Quantifizierung Seneszente 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. ...

Medizin

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.

Eine Quantitative Messung von reaktiven Sauerstoffspezies und Seneszenz-assoziierten sekretorischen Phänotyp in normalen menschlichen Fibroblasten während Onkogen-induzierte Seneszenz

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.

Simultane Bildgebung und Durchflusszytometrie-basierte Detektion mehrerer fluoreszierender Seneszenzmarker in therapieinduzierten seneszenten Krebszellen

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

Krebsforschung

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.

Far-Red Fluoreszenz-Seneszenz-assoziierte β-Galactosidase-Sonde zur Identifizierung und Anreicherung von seneszenten Tumorzellen mittels Durchflusszytometrie

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.

Messung von Proteinumsatzraten in seneszenten und nicht teilenden kultivierten Zellen mit metabolischer Markierung und Massenspektrometrie

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

Biochemie

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.

Techniken zur Induktion und Quantify Zelluläre Seneszenz

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 ...

Biologie

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.

SA--Galactosidase-basierter Screening-Test zur Identifizierung von Senotherapeutischen Arzneimitteln

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 ...