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

実りある議論のため管理者にラボと Thijmen ヴァン Vliet と紫外線による老化のプロトコルのデータを共有するためのメンバーに感謝いたします。

1. 一般準備
<ol><li>D10 メディアを準備します。10 %fbs と 1% ペニシリン/ストレプトマイシンを DMEM 培地 Glutamax を補う (最終濃度: 100 U/mL)。</li>
	<li>滅菌 PBS を準備します。製造元の指示に従って水の錠剤を溶解します。オートクレーブで滅菌します。</li>
	<li>1 x トリプシンを準備します。トリプシン Versene EDTA/10 の 5 mL の希釈 1:10 45 mL の滅菌 PBS で x。 注: プロトコルを通して我々 使用に近い?...

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

ここで説明したプロトコル最適化した一次線維芽細胞、特に BJ と WI-38 細胞。複製老化、電離放射線、ドキソルビシンのためのプロトコルは (HCA2 および IMR90) 線維芽細胞と他の細胞型 (すなわち新生児メラノサイトとケラチノ サイトまたは iPSC 由来心筋細胞) の他のタイプに正常に適用されています。本研究室では。ただし、その他の細胞のタイプのための適応は、シード細胞、メソッドおよ...

1961 年、ヘイフリック、ムーアヘッドは、文化の主要な線維芽細胞が連続通路1の後の増殖の可能性を失うことを報告します。このプロセスは、各細胞分裂後シーケンシャル テロメア短縮が原因です。テロメア批判的に短い長さに達する、増殖の不可逆的な停止を活性化する DNA 損傷応答 (DDR) によって認識されます-複製老化としても定義されています。複製老化細胞のマイトジェンとアポトーシス シグナル2,3の両方に依存しないレンダリングする永久的な細胞周期の停止の状態を誘導するために知られている多くの刺激の 1 つされて。老化プログラムは通常高リソソーム活動、ミトコンドリア、核変化、クロマチン再編成、小胞体ストレス、DNA の損傷と老化関連を含む追加の機能によって特徴付けられる分泌型表現型 (SASP)3,4。老化細胞体に複数の機能がある: 開発、創傷治癒およびがん抑制2。同様に、彼らは高齢化と、逆説的に、腫瘍の進行5で重要な役割を再生する知られています。老化の負の値、および部分的に矛盾した効果は、SASP6頻繁に原因です。
最近では、マウスから老化細胞の除去が寿命延長し老化機能7,8,9,10、11,の多くの除去につながることを示した12します。 同じ方法で複数の薬を老化細胞 (senolytics) を排除するか、または SASP13,14を対象に開発されています。アンチエイジング治療の可能性は、フィールドに多くの注目を集めている最近。
細胞の老化に関連したメカニズムの研究と薬理学的介入の上映前のヴィヴォモデルでは、特に主線維芽細胞上に依存します。老化の表現型の変化は観察と細胞の種類、刺激および時間ポイント3,15を含む様々 な要因に依存して多様な老化誘導因子によって活性化されるいくつかの一般的な機能がありますが、 16,17。勉強し、老化細胞をターゲットの不均一性を考慮することが不可欠です。したがって、このプロトコルは一連のさまざまな治療法を使用して、主に線維芽細胞老化を誘導するために使用されるメソッドを提供するために目指しています。それで説明しますが、メソッドは他の細胞型に適応できます。
別に複製老化老化を誘発する他の 5 つの治療について述べる: 電離放射線、紫外線 (UV)、ドキソルビシン、酸化ストレスおよびエピジェネティックな変化 (すなわち DNA 脱メチル化やヒストンのアセチル化の推進).電離放射線と紫外線の両方、直接 DNA 損傷を引き起こすし、適切な用量でトリガー老化18,19。ドキソルビシンまた間を DNA にインターカ レートによって DNA の損傷によって主に老化を引き起こすし、トポイソメラーゼ II の機能を中断、中止 DNA 修復機構20。老化に不可欠な遺伝子の発現は、DNA メチル化とヒストンのアセチル化によって制御されます通常。結果として、ヒストン脱アセチル化酵素阻害剤 (例えば、酪酸ナトリウム、サハ) と DNA demethylating (例えば5 字) エージェントは、正常細胞21,22老化をトリガーします。
最後に、4 つの老化細胞に関連付けられている最も一般的なマーカーの説明される: 老化関連-β-ガラクトシダーゼ (SA β gal) EdU 取り込みアッセイ、細胞周期調節因子の過剰発現による DNA 合成率の活動とサイクリン依存性キナーゼ阻害剤 p16 と p21 と、SASP のメンバーの分泌の過剰発現

<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

細胞の老化は、さまざまな有害な刺激への応答でアクティブに永久的な細胞周期の停止の状態です。細胞の老化の活性化は、腫瘍の抑制、組織改造と高齢化を含むさまざまな病態生理学的条件の特徴です。生体内で細胞老化の誘導はまだよく特徴付けられます。ただし、 ex vivo細胞老化を促進する刺激の数を使用できます。その中で、最も一般的な老化誘導が複製枯渇、電離放射線し非電離放射線、遺伝毒性薬、酸化ストレスと demethylating とエージェントをアセチル化します。ここでは、これらの刺激を使用して老化に線維芽細胞を誘導する方法で詳細な指示いたします。このプロトコルは、簡単に、初代培養細胞やがん細胞を含む細胞の種類ごとに適応できます。老化誘導の検証するための異なる方法についても述べる。特に、我々 はライソゾーム酵素老化関連 β ガラクトシダーゼ (SA β gal) 細胞周期の表現のレベル 5-エチニル-2'-デオキシウリジン (EdU) 取り込みアッセイを用いた DNA 合成率の活性の測定に焦点を当てるp16 の阻害剤と p21 と表情やメンバーの Senescence-Associated 分泌形質 (SASP) の分泌。最後に、私達は例の結果を提供し、これらのプロトコルのアプリケーションをさらに議論。

SA β gal 染色老化線維芽細胞の濃縮
Β-ガラクトシダーゼ (β gal) すべての細胞で発現していること、最適な pH 4.025,26のライソゾーム酵素であります。ただし、老化、中にリソソームのサイズが大きく、その結果、老化細胞蓄積 β gal。この酵素の増加量は、最適 pH 6.025</s...

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

ひと初代細胞における細胞老化の誘導性

ここでは、一連の誘発のためのプロトコルおよび培養細胞の細胞老化の検証について述べる。私たちはさまざまな老化を誘発する刺激に焦点を当てるし、一般的な老化関連マーカーの定量化を記述します。私たちはモデルとして線維芽細胞を使用して技術的な詳細を提供するが、プロトコルは、さまざまな携帯電話モデルに合わせることができます。

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

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