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

我们感谢 Demaria 实验室的成员进行了卓有成效的讨论, 并 Thijmen van 弗利特共享关于紫外线诱发衰老的数据和协议。

1. 一般准备
<ol><li>准备 D10 培养基。补充 DMEM 中 Glutamax 与10% 血清和1% 青霉素/链霉素 (最后浓度: 100 U/毫升)。</li>
	<li>准备无菌 PBS。根据制造商的指示, 在水中溶解药片。用高压釜消毒。</li>
	<li>准备1x 胰蛋白酶。稀释5毫升的胰蛋白酶-Versene EDTA/10x 1:10 在45毫升的无菌 PBS。 注意: 在整个协议中, 我们使用的细胞培养条件, 更接近的生理条件, 主要成纤维细胞。这意味着, 我们孵化的细胞在37摄?...

<ol>
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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. 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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. 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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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这里解释的协议被优化了为人的主要成纤维细胞, 特别是 BJ 和 WI-38 细胞。复制衰老, 电离辐射和阿霉素的协议已成功地应用于其他类型的成纤维细胞 (HCA2 和 IMR90) 和其他细胞类型 (即新生儿黑色素细胞和角质形成素或 iPSC 衍生心肌细胞)在我们的实验室里。然而, 可以通过调整一些细节, 如种子细胞数量, 方法和化学物质来帮助细胞附着/分离到塑料支架上, 以及治疗的剂量以避免毒性, 来优化其他细?...

在 1961年, 佛烈克和穆尔黑德报告说, 在连续1段后, 培养的主要成纤维细胞失去了增殖潜能。这一过程是由每细胞分裂后端粒的顺序缩短引起的。当端粒到达临界短的长度时, 它们被 DNA 损伤反应 (DDR) 所识别, 它激活了不可逆转的增殖--也被定义为复制衰老。复制衰老是目前已知的许多刺激之一, 导致一个永久性细胞周期逮捕的状态, 使细胞不敏感的 mitogens 和凋亡信号2,3。衰老程序通常的特点是额外的功能, 包括高溶酶活性, 线粒体功能障碍, 核变化, 染色质重组, 内质网应力, DNA 损伤和衰老相关分泌表型 (SASP)3,4。衰老细胞在体内具有多种功能: 发育、创面愈合和肿瘤抑制2。同样, 众所周知, 它们在衰老和肿瘤进展5中起着重要的作用。衰老的消极和部分矛盾的影响通常归因于 SASP6。
最近, 它表明, 消除衰老细胞的小鼠导致寿命延长和消除许多老化功能7,8,9,10,11,12. 同样, 已经开发了多种药物, 以消除衰老细胞 (senolytics) 或靶向 SASP13,14。抗衰老治疗潜力最近引起了人们的广泛关注。
对细胞衰老相关机制的研究和药理干预的筛选严重依赖于体外模型, 特别是在人原代成纤维细胞中。虽然不同的衰老诱导因子激活了一些共同的特征, 但衰老表型的大变异性被观察到, 并依赖于各种因素, 包括细胞类型, 刺激和时间点3,15, 16,17。研究和定位衰老细胞的异质性是当务之急。因此, 本议定书的目的是提供一系列方法, 以诱发衰老的主要成纤维细胞使用不同的治疗。正如它将解释, 这些方法可以很容易地适应其他细胞类型。
除了复制衰老, 我们还描述了其他五种衰老诱导治疗: 电离辐射, 紫外线 (紫外线) 辐射, 阿霉素, 氧化应激和后生变化 (即促进组蛋白乙酰化或 DNA 去甲基化).电离辐射和紫外线辐射直接导致 DNA 损伤, 并在适当剂量下引发衰老18,19。阿霉素还导致衰老主要是通过锂 dna 损伤的 dna 和干扰拓扑异构酶 II 功能, 从而停止 DNA 修复机制20。衰老的关键基因表达通常由组蛋白乙酰化和 DNA 甲基化控制。因此, 组蛋白乙酰抑制剂 (如丁酸钠和萨哈) 和 DNA 去甲基化 (例如, 5-aza) 剂会导致正常细胞21,22的衰老。
最后, 与衰老细胞相关的四种最常见的标记将被解释为: 衰老相关的乳糖酶 (SA β) 的活性、由免疫组织测定的 DNA 合成率、细胞周期调节器的过度表达和细胞周期蛋白依赖性激酶抑制剂 p16 和 p21, 并表达和分泌的 SASP 成员。

<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

细胞衰老是一种永久性细胞周期停止激活的反应, 以应对不同的破坏性刺激。细胞衰老的活化是各种病理生理条件的标志, 包括肿瘤抑制、组织重塑和衰老。细胞衰老的诱导体在体内仍然缺乏特征。然而, 一些刺激可以用来促进细胞衰老前体。其中, 最常见的衰老诱导因子是复制衰竭, 电离和非电离辐射, 毒性药物, 氧化应激, 去甲基化和乙酰剂。在这里, 我们将提供有关如何使用这些刺激诱导成纤维细胞衰老的详细说明。该协议可以很容易地适应不同类型的主要细胞和细胞系, 包括癌细胞。我们还描述了不同的方法来验证衰老诱导。特别是, 我们专注于测量溶酶体与衰老相关的β-乳糖酶 (SA β-加仑) 的活性, 使用 5-乙炔-2 '-脱氧尿苷 () 的 DNA 合成率, 细胞周期的表达水平抑制剂 p16 和 p21, 以及衰老相关分泌表型 (SASP) 成员的表达和分泌。最后, 给出了实例结果, 并讨论了这些协议的进一步应用。

衰老成纤维细胞中 SA β-gal 染色的富集
β-乳糖酶 (β-加仑) 是一种酶, 表达在所有细胞和具有最佳 pH 值为 4.025,26。然而, 在衰老过程中, 溶酶体体积增大, 衰老细胞积累β-加仑。这种酶的增加使它有可能检测到它的活性, 即使在次优 pH 值 6.025,27?...

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

原发性细胞衰老的诱导与鉴定

在这里, 我们讨论了一系列的诱导和确认细胞衰老在培养细胞的协议。我们专注于不同的衰老诱发刺激, 并描述了常见的衰老相关标记的量化。我们提供的技术细节, 以成纤维细胞作为模型, 但协议可以适应各种细胞模型。

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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Cell Proliferation

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