Name: a sensitive method to quantify senescent cancer cells
Uploaded: 2013-08-02T13:00:00
Description: Watch this Scientific Journal Video about A Sensitive Method to Quantify Senescent Cancer Cells at JoVE.com

我们感谢ICORE SF 4206（卡昂大学）为利用流式细胞设施。 JC收到博士后奖学金委员会辐射防护EDF和下诺曼底地区行政法院。这些数据国家法甲抗癌症 - ComitésDE L&#39;奥恩等卡尔瓦多斯资助项目的一部分。

1。巴弗洛霉素A1，C12FDG和细胞培养制备<ol><li> C 12的溶解在二甲亚砜（DMSO）中至最终浓度为20mM的葡萄糖粉。分装和储存在-20°C DMSO应采用适当的安全条件。 </li><li>巴弗洛霉素A1粉溶解在DMSO中，使最终浓度为0.1mM。分装和储存在 - 20°C。巴弗洛霉素应采用适当的安全条件。 </li><li> RPMI 8226细胞系，或其他细胞系粘附或不培养，在含有10％小牛血清和1％的抗生素，在37℃，5％CO 2的介?...

<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>Campisi, J., d'Adda di Fagagna, F. <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:+when+bad+things+happen+to+good+cells.">Cellular senescence: when bad things happen to good cells.</a> Nature Review Molecular Cell Biology. 8 (9), 729-740 (2007).</li><li>Shay, J. W., Roninson, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+senescence+in+carcinogenesis+and+cancer+therapy.">Hallmarks of senescence in carcinogenesis and cancer therapy.</a> Oncogene. 23 (16), 2919-2933 (2004).</li><li>Bartkova, J., Rezaei, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogene-induced+senescence+is+part+of+the+tumorigenesis+barrier+imposed+by+DNA+damage+checkpoints.">Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints.</a> Nature. 444 (7119), 633-637 (2006).</li><li>Takacova, S., Slany, R., Bartkova, J., Stranecky, V., Dolezel, P., Luzna, 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=DNA+damage+response+and+inflammatory+signaling+limit+the+MLL-ENL-induced+leukemogenesis+in+vivo.">DNA damage response and inflammatory signaling limit the MLL-ENL-induced leukemogenesis in vivo.</a> Cancer Cell. 21, 517-531 (2012).</li><li>Krtolica, A., Parrinello, S., Lockett, S., Desprez, P. Y., 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=Senescent+fibroblasts+promote+epithelial+cell+growth+and+tumorigenesis:+a+link+between+cancer+and+aging.">Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging.</a> Proceedings of the National Academy of Sciences. 98, 12072-12077 (2001).</li><li>Coppe, J. P., Patil, C. K., Rodier, F., Sun, Y., Munoz, D. P., Goldstein, 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=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, 2853-2868 (2008).</li><li>Roninson, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+senescence+in+cancer+treatment.">Tumor cell senescence in cancer treatment.</a> Cancer Research. 63 (11), 2705-2715 (2003).</li><li>Dimri, G. P., Lee, X., 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), 9363-937 (1995).</li><li>Kurz, D. J., Decary, S., Hong, Y., Erusalimsky, J. D. <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+(beta)-galactosidase+reflects+an+increase+in+lysosomal+mass+during+replicative+ageing+of+human+endothelial+cells.">Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells.</a> Journal of Cell Science. 113, 3613-3622 (2000).</li><li>Debacq-Chainiaux, F., Erusalimsky, J. D., Campisi, J., Toussaint, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+to+detect+senescence-associated+beta-galactosidase+(SA-betagal)+activity,+a+biomarker+of+senescent+cells+in+culture+and+in+vivo.">Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo.</a> Nature Protocols. 4 (12), 1798-1806 (2009).</li><li>Cahu, J., Bustany, S., Sola, B. <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+phenotype+favors+the+emergence+of+cancer+stem-like+cells.">Senescence-associated secretory phenotype favors the emergence of cancer stem-like cells.</a> Cell Death and Disease. Dec 20, 3-e446 (2012).</li></ol>

这里，我们介绍一个基于流式细胞仪的方法来量化在肝癌细胞中的细胞衰老。这种方法是基于使用的膜透过性化合物中，C 12 FDG，因此，可以使用任何类型的细胞，并经过各种治疗方法，只要是采取了由细胞的化合物。细胞摄取后，C 12 FDG是由β-半乳糖苷酶，富含衰老细胞的溶酶体水解。激光激发后，化合物发出绿色荧光，使衰老细胞的定量。这种量化的绿色荧光可以通过流式细?...

海弗利克和穆尔黑德自六十年代初，它的发现，衰老仍然蜂窝的好奇心1。人体细胞不会无限增殖，衰老，海弗利克和穆尔黑德创造了细胞老化过程。衰老是一个稳定的细胞中，细胞周期阻滞保持代谢活跃，而不​​是细胞死亡。迄今为止，已经示出了4个细胞机制，诱导衰老：端粒缩短，DNA损伤，染色质的扰动，促有丝分裂原癌基因的异常表达2。这表明，不仅是正常的细胞，而且还能够接受衰老3癌细胞。事实上，癌症细胞衰老示出了进一步的肿瘤进展4-5构成障碍。然而，一些报告已经指出，在某些情况下，细胞衰老也可以促进恶性6-7。研究衰老癌症CELls是在癌症研究中，目前使用的化疗药物可导致癌细胞不仅在体外还是在体内 8衰老成为一种冲动。 主站的检测，以探讨衰老细胞的存在下，在酸性pH下的β-半乳糖苷酶测定。该组织化学法反映在溶酶体发生在大多数衰老细胞的生物合成增加。简单地说，外源X-gal的，适用于细胞，由衰老细胞的溶酶体中丰富的β-半乳糖苷酶裂解，从而产生蓝色9。蓝色衰老细胞，然后可以量化明场显微镜下。虽然很受欢迎，β-半乳糖苷酶检测提出了主要的缺点。首先，这对固定的细胞进行测定。第二，它是耗时的，因为它意味着衰老程度的量化，通过计算衰老细胞在显微镜下的一个显着高。第三，该法不敏感，衰老和非衰老细胞之间的歧视完全依赖于观察者。 我们在这里讨论的未开发，快速，敏感的流式细胞仪检测，以评估现场衰老细胞的存在。此法是基于水解的膜渗透分子的5 dodecanoylaminofluorescein的二-β-D-吡喃半乳糖苷（C 12 FDG），β-半乳糖苷酶在衰老细胞中富集。水解和激光激发后，在C 12 FDG发出绿色荧光，因此，可以通过流式细胞仪10，11检测到。衰老的细胞通过流式细胞仪的检测提供了快速，灵敏的巨大优势。此外，通过流式细胞术检测衰老细胞与其他细胞的标记物的检测可以耦合。本试剂盒可用于检测衰老细胞，用化疗治疗的癌细胞内的人口，可以常规设置组织切片，细胞分离后，在诊所。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the Reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>5-dodecanoylaminofluorescein di-&beta;-D-galactopyranoside </td>
			<td>Invitrogen</td>
			<td>D-2893</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dimethyl sulfoxide </td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Bafilomycin A1</td>
			<td>Sigma</td>
			<td>B1793</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Doxorubicin</td>
			<td>Sandoz</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypan blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		 </tr>
		 <tr>
			<td>RPMI 1640 cell culture medium</td>
			<td>Lonza</td>
			<td>BE12-702</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal calf serum</td>
			<td>PAA Laboratories GmBH</td>
			<td>A15 101</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Antibiotics</td>
			<td>Gibco</td>
			<td>5290-018</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gallios flow cytometer</td>
			<td>Beckman Coulter</td>
			<td>A94291</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

a sensitive method to quantify senescent cancer cells

人体细胞不会无限增殖。外部和/或内在线索后，细胞可能会死，或进入一个稳定的细胞周期阻滞，称为衰老。几个细胞的机制，如端粒缩短和有丝分裂原癌基因的异常表达，已被证明是导致衰老。衰老并不局限于对正常细胞也有报道癌细胞衰老。 化学治疗药物已被证明在肿瘤细胞中诱导衰老。但是，它仍然是有争议的衰老是否阻止或促进肿瘤的发生。因为它最终可能特定病人，一种快速，灵敏的方法将很快被要求评估在癌症细胞的衰老。 的是标准的β-半乳糖苷酶的测定中，目前使用的方法，为此，提出了主要的缺点：它是费时和不敏感。在这里我们提出一个基于流式细胞仪检测，以研究衰老对李VE细胞。本试剂盒提供的优点是快速，灵敏，可耦合到各种细胞标记物的免疫标记。

多发性骨髓瘤，血液肿瘤，被用来评估的过程化学治疗诱导的衰老。要做到这一点市售MM细胞株（RPMI 8226）治疗与化学治疗药物阿霉素，2天。然后将细胞进行治疗巴弗洛霉素A1和C 12 FDG进一步培养。细胞，通过流式细胞术分析， 图1说明了不同的步骤和衰老细胞的百分比可确定在一天之内使用的C 12 FDG。首先运行未标记细胞在流式细胞仪。 如图2A中所示，一...

Watch this Scientific Journal Video about A Sensitive Method to Quantify Senescent Cancer Cells at JoVE.com

A Sensitive Method to Quantify Senescent Cancer Cells

无论是衰老阻止或促进肿瘤的发生仍存在争议。由于化学治疗药物可以诱导癌细胞衰老，衰老研究提出的新疗法是必不可少的。然而，标准的和广泛使用的β-半乳糖苷酶测定的主要缺点。在这里我们提出一种快速，灵敏的基于流式细胞仪检测，以量化衰老。

量化衰老癌症细胞的敏感方法

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

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