Name: far-red fluorescent senescence-associated &#946;-galactosidase probe for identification and enrichment of senescent tumor cells by flow cytometry
Uploaded: 2022-09-13T13:40:00
Description: Watch this Scientific Journal Video about Far-Red Fluorescent Senescence-Associated ÃŽÂ²-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry at JoVE.com

我们感谢芝加哥大学的细胞术和抗体核心设施对流式细胞术仪器的支持。芝加哥大学的动物研究中心提供动物住房。

所描述的所有动物工作都得到了芝加哥大学机构动物护理和使用委员会的批准。
1. 储备溶液的制备和储存
注意：如果要对细胞进行流动分选，则应使用无菌技术制备所有溶液，并通过0.22μm过滤装置过滤。
<ol><li>在DMSO中制备5 mg / mL的DDAO-半乳糖苷储备溶液。每管 50 μL（或所需体积）等分。在黑暗中在-20°C下储存长达1年。</li>
	<li>在DMSO中以1m...

<ol>
	<li>Saleh, T., Tyutyunyk-Massey, L., Gewirtz, D. A. <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+escape+from+therapy-induced+senescence+as+a+model+of+disease+recurrence+after+dormancy.">Tumor cell escape from therapy-induced senescence as a model of disease recurrence after dormancy.</a> Cancer Research. 79 (6), 1044-1046 (2019).</li><li>Wang, B., Kohli, J., 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=Senescent+cells+in+cancer+therapy:+friends+or+foes.">Senescent cells in cancer therapy: friends or foes.</a> Trends in Cancer. 6 (10), 838-857 (2020).</li><li>Prasanna, P. 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L., 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+in+cancer:+from+mechanisms+to+detection.">Cellular senescence in cancer: from mechanisms to detection.</a> Molecular Oncology. 15 (10), 2634-2671 (2021).</li><li>Hernandez-Segura, A., Nehme, J., 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=Hallmarks+of+cellular+senescence.">Hallmarks of cellular senescence.</a> Trends in Cell Biology. 28 (6), 436-453 (2018).</li><li>Bojko, A., Czarnecka-Herok, J., Charzynska, A., Dabrowski, M., Sikora, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+the+senescence+phenotype+of+cancer+cells+treated+with+chemotherapeutic+agents.">Diversity of the senescence phenotype of cancer cells treated with chemotherapeutic agents.</a> Cells. 8 (12), 1501 (2019).</li><li>Miku&#322;a-Pietrasik, J., Niklas, A., Uruski, P., Tykarski, A., Ksi&#261;&#380;ek, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+significance+of+therapy-induced+and+spontaneous+senescence+of+cancer+cells.">Mechanisms and significance of therapy-induced and spontaneous senescence of cancer cells.</a> Cellular and Molecular Life Sciences. 77 (2), 213-229 (2020).</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+beta-galactosidase+is+lysosomal+beta-galactosidase.">Senescence-associated beta-galactosidase is lysosomal beta-galactosidase.</a> Aging cell. 5 (2), 187-195 (2006).</li><li>Itahana, K., Itahana, Y., Dimri, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+detection+of+senescence-associated+&#946;+galactosidase.">Colorimetric detection of senescence-associated &#946; galactosidase.</a> Methods in Molecular Biology. 965, 143-156 (2013).</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 of the United States of America. 92 (20), 9363-9367 (1995).</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>Noppe, 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=Rapid+flow+cytometric+method+for+measuring+senescence+associated+beta-galactosidase+activity+in+human+fibroblasts.">Rapid flow cytometric method for measuring senescence associated beta-galactosidase activity in human fibroblasts.</a> Cytometry A. 75 (11), 910-916 (2009).</li><li>Tung, C. -. 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=In+vivo+imaging+of+&#946;-galactosidase+activity+using+far+red+fluorescent+switch.">In vivo imaging of &#946;-galactosidase activity using far red fluorescent switch.</a> Cancer Research. 64 (5), 1579-1583 (2004).</li><li>Gong, 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=beta-Galactosidase+activity+assay+using+far-red-shifted+fluorescent+substrate+DDAOG.">beta-Galactosidase activity assay using far-red-shifted fluorescent substrate DDAOG.</a> Analytical Biochemistry. 386 (1), 59-64 (2009).</li><li>Terman, A., Brunk, U. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipofuscin:+Mechanisms+of+formation+and+increase+with+age.">Lipofuscin: Mechanisms of formation and increase with age.</a> APMIS. 106 (2), 265-276 (1998).</li><li>Georgakopoulou, E. 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=Specific+lipofuscin+staining+as+a+novel+biomarker+to+detect+replicative+and+stress-induced+senescence.+A+method+applicable+in+cryo-preserved+and+archival+tissues.">Specific lipofuscin staining as a novel biomarker to detect replicative and stress-induced senescence. A method applicable in cryo-preserved and archival tissues.</a> Aging. 5 (1), 37-50 (2013).</li><li>Wang, B., 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=The+quest+to+define+and+target+cellular+senescence+in+cancer.">The quest to define and target cellular senescence in cancer.</a> Cancer Research. 81 (24), 6087-6089 (2021).</li><li>Appelbe, O. K., Zhang, Q., Pelizzari, C. A., Weichselbaum, R. R., Kron, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-guided+radiotherapy+targets+macromolecules+through+altering+the+tumor+microenvironment.">Image-guided radiotherapy targets macromolecules through altering the tumor microenvironment.</a> Molecular Pharmaceutics. 13 (10), 3457-3467 (2016).</li><li>Maciorowski, Z., Chattopadhyay, P. 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在过去十年左右的时间里，由于肿瘤免疫学的日益普及，低成本流式细胞仪的发展以及学术机构共享仪器设施的改进，流式细胞术已成为癌症研究中更常见的分析平台。多色检测现在是标准配置，因为大多数较新的仪器都配备了紫色、蓝绿色和红色至远红色的光学阵列。因此，该DDAOG方案可能与各种流式细胞仪兼容。当然，任何流式细胞仪都应经过用户评估。在DDAOG测定中添加额外的荧光团（例如...

衰老细胞通常在正常的生物衰老过程中在生物体内积累多年，但也可能在肿瘤细胞中迅速发展，作为对各种癌症治疗（包括放疗和化疗）引起的损伤的反应。虽然不再增殖，但治疗诱导的衰老（TIS）肿瘤细胞可能有助于治疗耐药性并导致复发1，2，3。TIS细胞分泌的因子可以通过促进免疫逃避或转移来加剧肿瘤恶性肿瘤4，5。TIS细胞产生复杂的，上下文特异性表型，改变的代谢谱和独特的免疫反应6，7，8。因此，各种癌症治疗方法诱导的TIS肿瘤细胞的鉴定和表征是癌症研究界持续关注的课题。
为了检测TIS肿瘤细胞，常规衰老测定被广泛使用，主要基于检测衰老标志酶溶酶体β-半乳糖苷酶GLB19的活性增加。在接近中性（而不是酸性）溶酶体 pH 下检测可以特异性检测衰老相关的 β-半乳糖苷酶 （SA-β-Gal）10。已经使用了几十年的标准SA-β-Gal测定使用X-Gal（5-溴-4-氯-3-吲哚基-β-D-吡喃半乳糖苷），一种蓝色显色β-半乳糖苷底物，通过光学显微镜检测固定细胞中的SA-β-Gal11。X-Gal 测定允许使用常用试剂和实验室设备对 TIS 进行定性视觉确认。基本的透射光显微镜是评估蓝色显色原存在所需的唯一仪器。然而，X-Gal 染色程序可能缺乏敏感性，有时需要超过 24 小时才能显色。染色之后，根据在光学显微镜下对表现出一定强度的蓝色色原的细胞进行计数，对单个衰老细胞进行低通量主观评分。由于X-Gal是不可渗透的，因此该测定需要溶剂固定的细胞，这些细胞无法回收用于下游分析。当处理来自动物或患者的有限样本时，这可能是一个主要缺点。
使用细胞通透性荧光酶底物改进的 SA-β-Gal 测定，包括 C 12-FDG（5-十二烷基氨基荧光素 Di-β-D-吡喃半乳糖苷，绿色）和 DDAOG（9H-（1，3-二氯-9，9-二甲基吖啶-2-酮-7-基）β-D-吡喃半乳糖苷，远红色）先前已出现在文献12，13，14，15 中。DDAOG的化学探针结构和光学特性见补充图S1。这些细胞通透性探针允许分析活细胞（而不是固定细胞），荧光探针而不是显色探针有助于使用快速高通量荧光分析平台，包括高内涵筛选仪器和流式细胞仪。分选流式细胞仪能够从细胞培养物或肿瘤中回收富集的活衰老细胞群，以进行下游分析（例如，蛋白质印迹、ELISA或“组学”。荧光分析还提供定量信号，可以更准确地确定给定样品中衰老细胞的百分比。可以轻松添加其他荧光探针，包括活性探针和荧光团标记的抗体，用于SA-β-Gal以外的靶标的多重分析。
与DDAOG类似，C12-FDG是SA-β-Gal的荧光探针，但其绿色荧光发射与内在细胞AF重叠，后者在衰老期间由于细胞16中脂褐素聚集体的积累而产生。通过使用远红DDAOG探针，绿色细胞AF可以用作确认衰老的辅助参数17。这通过在SA-β-Gal之外使用第二个标记物来提高测定可靠性，而SA--Gal作为衰老的单一标记物通常不可靠18。由于检测衰老细胞中的内源性AF是一种无标记的方法，因此它是扩展我们基于DDAOG的测定特异性的快速简便方法。
在该协议中，我们证明了使用DDAOG和AF作为快速，双参数流式细胞术测定，用于鉴定来自 体外 培养物或从小鼠中建立的药物治疗肿瘤中分离的活TIS肿瘤细胞（图1）。该方案使用与各种标准商业流式细胞仪和分选仪兼容的荧光团（表1）。使用标准流式细胞术分析可以定量活衰老细胞的百分比。如果需要，可以执行可选的免疫标记步骤以评估与衰老同时感兴趣的细胞表面抗原。鉴定的衰老细胞也可以使用标准的荧光激活细胞分选（FACS）方法进行富集。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64176/64176fig01.jpg"> 图 1：实验性工作流程。 总结DDAOG测定关键点的示意图。（A）将TIS诱导药物添加到哺乳动物培养的细胞中或施用于荷瘤小鼠。然后允许时间用于TIS的发作：对于细胞，治疗后4天;对于小鼠，总共22天，每5天进行三次治疗，外加7天恢复。收获细胞或将肿瘤解离成悬浮液。（B）用Baf处理样品以调节溶酶体pH以检测SA-β-Gal30分钟;然后，加入DDAOG探针60分钟以检测SA-β-Gal。将样品在PBS中洗涤2次，并短暂添加活性染色剂（15分钟）。或者，样品可以在开放的荧光通道中用荧光抗体染色和/或固定以供以后分析。（C）使用标准流式细胞仪分析样品。活细胞在点图中可视化，显示红色DDAOG（指示SA-β-Gal）与绿色自发荧光（脂褐素）。基于未处理的对照样品（未显示）建立确定TIS细胞百分比的门。如果使用分选细胞仪（FACS），则可以收集TIS细胞并将其放回培养物中以进行进一步的 体外 测定，或者裂解并处理以进行分子生物学测定。缩写：DDAO = 9H-（1，3-二氯-9，9-二甲基吖啶-2-酮）;DDAOG = DDAO-半乳糖苷;TIS=治疗诱导的衰老;FL-Ab = 荧光团偶联抗体;Baf = 巴非霉素 A1;SA-β-Gal = 衰老相关的 β-半乳糖苷酶;PBS = 磷酸盐缓冲盐水;FACS = 荧光激活细胞分选。 <a href="https://www.jove.com/files/ftp_upload/64176/64176fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>荧光基团</td>
			<td>检测</td>
			<td>防爆/电磁（纳米）</td>
			<td>激光细胞仪（纳米）</td>
			<td>细胞仪检测器/带通滤光片（纳米）</td>
		</tr><tr><td>东道格</td>
			<td>SA-β-加尔</td>
			<td>645/6601</td>
			<td>640</td>
			<td>670 / 30</td>
		</tr><tr><td>自动对焦</td>
			<td>脂褐素</td>
			<td>< 600</td>
			<td>488</td>
			<td>525 / 50</td>
		</tr><tr><td>CV450</td>
			<td>可行性</td>
			<td>408/450</td>
			<td>405</td>
			<td>450 / 50</td>
		</tr><tr><td>体育</td>
			<td>抗体/表面标志物</td>
			<td>565/578</td>
			<td>561</td>
			<td>582 / 15</td>
		</tr></tbody></table>表 1：荧光团和细胞仪光学规格。 本协议中使用的细胞仪规格列出了总共具有 4 个激光器和 15 个发射检测器的仪器。在 645/660 nm 处检测到的 DDAOG 是被 SA-β-Gal1 切割的探针的形式。未裂解的DDAOG可以在460/610nm处表现出低水平的荧光，但通过方案中的洗涤步骤去除。缩写：DDAO = 9H-（1，3-二氯-9，9-二甲基吖啶-2-酮）;DDAOG = DDAO-半乳糖苷;AF = 自发荧光;PE = 藻红蛋白;SA-β-Gal = 衰老相关的 β-半乳糖苷酶。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bafilomycin A1</td>
			<td>Research Products International</td>
			<td>B40500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleomycin sulfate&#160;</td>
			<td>Cayman</td>
			<td>13877</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>US Biological</td>
			<td>A1380</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein Violet 450 AM viability dye</td>
			<td>ThermoFisher Scientific</td>
			<td>65-0854-39</td>
			<td>eBioscience</td>
		</tr>
		<tr>
			<td>DPP4 antibody, PE conjugate</td>
			<td>Biolegend</td>
			<td>137803</td>
			<td>Clone H194-112</td>
		</tr>
		<tr>
			<td>Cell line: A549 human lung adenocarcinoma</td>
			<td>American Type Culture Collection</td>
			<td>CCL-185</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell line: B16-F10 mouse melanoma</td>
			<td>American Type Culture Collection</td>
			<td>CRL-6475</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Corning</td>
			<td>3008</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainers, 100 &#181;m</td>
			<td>Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>DDAO-Galactoside</td>
			<td>Life Technologies</td>
			<td>D6488</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM medium 1x</td>
			<td>Life Technologies</td>
			<td>11960-069</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Sigma</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxorubicin, hydrochloride injection (USP)</td>
			<td>Pfizer</td>
			<td>NDC 0069-3032-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxorubicin, PEGylated liposomal (USP)</td>
			<td>Sun Pharmaceutical</td>
			<td>NDC 47335-049-40</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA 0.5 M</td>
			<td>Life Technologies</td>
			<td>15575-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Etoposide&#160;</td>
			<td>Cayman</td>
			<td>12092</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Omega&#160;</td>
			<td>FB-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc receptor blocking reagent</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td>Anti-mouse CD16/32</td>
		</tr>
		<tr>
			<td>Flow cytometer (cell analyzer)</td>
			<td>Becton Dickinson (BD)</td>
			<td>Various</td>
			<td>LSRFortessa</td>
		</tr>
		<tr>
			<td>Flow cytometer (cell sorter)</td>
			<td>Becton Dickinson (BD)</td>
			<td>Various</td>
			<td>FACSAria</td>
		</tr>
		<tr>
			<td>GlutaMax 100x</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1 M</td>
			<td>Lonza</td>
			<td>BW17737</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase TL</td>
			<td>Sigma</td>
			<td>5401020001</td>
			<td>Roche</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 100x</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) 1x</td>
			<td>Corning</td>
			<td>MT21031CV</td>
			<td>Dulbecco&#39;s PBS (without calcium and magnesium)</td>
		</tr>
		<tr>
			<td>Rainbow calibration particles, ultra kit</td>
			<td>SpheroTech</td>
			<td>UCRP-38-2K</td>
			<td>3.5-3.9 &#181;m, 2E6/mL</td>
		</tr>
		<tr>
			<td>RPMI-1640 medium 1x</td>
			<td>Life Technologies</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride 0.9% (USP)</td>
			<td>Baxter Healthcare Corporation</td>
			<td>2B1324</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for cytometer data acquisition, &#34;FACSDiva&#34;</td>
			<td>Becton Dickinson (BD)</td>
			<td>n/a</td>
			<td>Contact BD for license</td>
		</tr>
		<tr>
			<td>Software for cytometer data analysis, &#34;FlowJo&#34;</td>
			<td>TreeStar</td>
			<td>n/a</td>
			<td>Contact TreeStar for license</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25%</td>
			<td>Life Technologies</td>
			<td>25200-114</td>
			<td></td>
		</tr>
	</tbody>
</table>

far-red fluorescent senescence-associated &#946;-galactosidase probe for identification and enrichment of senescent tumor cells by flow cytometry

细胞衰老是由生物损伤引起的增殖性停滞状态，通常在衰老细胞中累积多年，但也可能在肿瘤细胞中迅速出现，作为对各种癌症治疗诱导的损伤的反应。肿瘤细胞衰老通常被认为是不可取的，因为衰老细胞对死亡产生抵抗力并阻止肿瘤缓解，同时加剧肿瘤恶性肿瘤和治疗耐药性。因此，衰老肿瘤细胞的鉴定是癌症研究界持续关注的问题。存在各种衰老测定，许多基于众所周知的衰老标志物，衰老相关β-半乳糖苷酶（SA-β-Gal）的活性。 
通常，SA-β-Gal测定是在固定细胞上使用显色底物（X-Gal）进行的，通过光学显微镜缓慢而主观地计数“蓝色”衰老细胞。使用细胞渗透性荧光SA-β-Gal底物（包括C12-FDG（绿色）和DDAO-半乳糖苷（DDAOG;远红色））的改进检测能够分析活细胞，并允许使用高通量荧光分析平台，包括流式细胞仪。C12-FDG是SA-β-Gal的有据可查的探针，但其绿色荧光发射与衰老期间由于脂褐素聚集体的积累而产生的固有细胞自发荧光（AF）重叠。通过使用远红SA-β-Gal探针DDAOG，绿色细胞自发荧光可用作确认衰老的次要参数，从而增加测定的可靠性。其余荧光通道可用于细胞活力染色或可选的荧光免疫标记。
使用流式细胞术，我们证明了使用DDAOG和脂褐素自发荧光作为双参数测定来鉴定衰老肿瘤细胞。进行活衰老细胞百分比的定量。如果需要，可以包括可选的免疫标记步骤来评估感兴趣的细胞表面抗原。鉴定出的衰老细胞也可以进行流式细胞术分选和收集以进行下游分析。收集的衰老细胞可以立即裂解（例如，用于免疫测定或“组学分析”）或进一步培养。

进行了几个实验，以证明DDAOG与X-Gal和C12-FDG在SA-β-Gal检测衰老方面的可比性。首先，X-Gal用于染色由ETO诱导的衰老B16-F10黑色素瘤细胞（图2A）。在ETO处理的细胞亚群中出现强烈的蓝色，而其他细胞表现出不太强烈的蓝色染色。大多数ETO处理的细胞的形态扩大。用荧光SA-β-Gal底物C12-FDG（绿色）或DDAOG（远红）染色ETO处理的细胞显示出与X-Gal相当的染色模式和强度...

Watch this Scientific Journal Video about Far-Red Fluorescent Senescence-Associated ÃŽÂ²-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry at JoVE.com

Far-Red Fluorescent Senescence-Associated ÃƒÅ½Ã‚Â²-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

提出了一种荧光流式细胞术定量由细胞培养或小鼠肿瘤模型中化疗药物诱导的衰老癌细胞的方案。可选程序包括共免疫染色、便于大批量或时间点分析的样品固定，以及通过流式细胞术分选富集活衰老细胞。

远红荧光衰老相关β-半乳糖苷酶探针，用于通过流式细胞术鉴定和富集衰老肿瘤细胞

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge ...

我们的多参数流式细胞术方案能够快速和改进地检测肿瘤细胞中治疗诱导的衰老，这是癌症生物学和治疗中正在进行的研究条件。我们的流式细胞术衰老检测比现有的基于显微镜的检测更快。我们使用两个标记物，而不仅仅是一个，来识别衰老细胞，提高测定可靠性。细胞可以与荧光抗体共染色，以检测其他感兴趣的靶标。流式细胞术分选可用于富集衰老细胞群以进行下游分析。该测定可以检测培养的癌细胞系中的衰老细胞，或在温和解离后的整个肿瘤样品中。在尝试这种技术之前，熟悉所使用的流式细胞仪的一般操作程序非常重要。有关推荐的流式细胞仪规格，请参阅文本。在药物诱导衰老前一天，用0.25%胰蛋白酶EDTA收获癌细胞系培养物。将细胞置于37摄氏度下五分钟以激活胰蛋白酶，并分离细胞单层。当单层明显脱落时，通过加入等体积的完全培养基来中和胰蛋白酶。将细胞悬液转移到无菌锥形管中。使用标准血细胞计数器方法计数细胞，并记录每毫升细胞数。在标准的六孔塑料培养板中以每平方厘米10至3至10倍10至第三细胞的速度板细胞。将板在 37 摄氏度、5% 二氧化碳和湿度下孵育过夜。第二天，用感兴趣的衰老诱导剂（如依托泊苷）处理细胞。然后孵育四天以允许衰老开始。每天在光学显微镜下检查细胞，以了解预期的形态变化。衰老开始后，通过在37摄氏度下添加胰蛋白酶EDTA五分钟来收获细胞。当细胞解离成悬浮液时，用等体积的完全培养基中和胰蛋白酶。将每个细胞悬液孔转移到1.7毫升微量离心管中。再次计数细胞并将每个样品等分到一组新的试管中。将试管在4摄氏度下以1， 000g离心5分钟，然后除去上清液。首先，通过将DMEM中一微摩尔巴非霉素A的溶液以每毫升1乘以10至第六个细胞的浓度添加到细胞沉淀中，并移液混合来调节培养细胞样品的溶酶体pH值。在 37 摄氏度的旋转器上以慢速孵育 30 分钟。然后为了染色衰老相关的β-半乳糖苷酶，在不洗涤的情况下以每毫升10微克的速度向样品中加入DDAOG储备溶液。放在旋转器上 60 分钟，避免光线直射。像以前一样，离心管，除去上清液，并在PBS中用一毫升冰冷的0.5%BSA洗涤沉淀。然后将300微升稀释的钙黄绿素紫450AM溶液加入洗涤的细胞沉淀中。在黑暗中在冰上孵育15分钟。将细胞样品转移到流式细胞仪兼容的试管中。在数据采集软件中，打开紫色通道直方图和远红色通道与绿色通道点图。以低进样速度启动细胞仪数据采集，并将用DDAOG染色的阳性对照样品放在进样口。开始获取样本数据。调整通道电压，使每个图中包含90%以上的事件。当所有设置都最佳时，每个样本记录 10， 000 个事件。将沾有DDAOG的车辆专用对照样品放在进气口上。启动数据采集并为每个样本记录 10， 000 个事件。寻找自发荧光或 AF 和 DDAO-半乳糖苷信号与阳性对照的升高。将样品数据保存为fcs文件格式，并将文件导出到配备流式细胞术分析软件的工作站计算机。使用细胞术数据分析软件，加载所有采集样品的 fcs 数据文件。首先，要对活细胞进行门控，请双击仅车辆控件的示例数据以打开其数据窗口。将数据可视化为紫色通道直方图。用CV 450染色的活细胞基于其比死细胞更亮的荧光。使用单门直方图工具绘制门以仅包含活细胞。将门命名为可行。然后将“可行门”从示例布局窗口拖到其他单元格样本上，以均匀应用门。打开布局窗口。在布局窗口中，将所有样本可视化为紫色通道直方图。验证活细胞门控是否适合所有样品。如果没有，请根据需要进行调整。接下来，要对衰老细胞进行门控，请双击仅车辆控件的门控活细胞数据以打开其数据窗口。然后将数据可视化为远红色通道与绿色通道的点图。使用矩形门控工具绘制门，以包含右上象限中少于 5% 的 DDAO 阳性和 AF 阳性细胞。将大门命名为Senescent。然后从示例布局窗口中将 Senescent 门拖动到所有样本的可行子集上，以均匀应用门。在布局窗口中，拖放所有可行的单元门控子集。将所有可行样本可视化为远红色与绿色通道点图，并观察结果。确保衰老门在所有图上都可见，并且仅车辆控制的门显示小于5%的衰老细胞。分析现已完成。现在可以根据需要以图形和表格格式导出数据。与未处理的细胞相比，依托泊苷诱导的衰老B16-F10黑色素瘤细胞由于衰老相关β-半乳糖苷酶升高切割X-gal而表现出扩大的形态和蓝色染色。用荧光C12FDG或DDAOG对依托泊苷处理的细胞进行染色显示出与X-gal相当的染色模式和强度变化。然而，与绿色C12FDG发射重叠的细胞自发荧光在未染色的衰老细胞中积聚。相比之下，自发荧光在DDAOG的远红光发射范围内通常可以忽略不计。此处显示了散点图的流式细胞仪数据采集设置、五峰商用彩虹荧光校准微球以及染色细胞的单通道荧光数据。DDAOG流式细胞仪B16-F10黑色素瘤细胞衰老测定数据显示，依托泊苷诱导的35%活细胞发生治疗诱导的衰老（TIS），增肽剂ABT-263几乎消除了TIS细胞。在A549肺癌细胞中，博来霉素诱导的TIS在66%的活细胞和ABT-263中将百分比降低到15。单独的ABT-263对未经处理的增殖细胞没有毒性。在抗体共染色测定中，PE 通道数据的直方图显示，42% 的依托泊苷处理的细胞对衰老标志物 DPPP4 阳性呈阳性。此外，二维点图的可视化表明，44%的依托泊苷处理的细胞对DDAOG和DPP4呈双阳性，而仅载体细胞为4%。接下来评估DDAOG染色细胞的固定。与未固定的对照样品相比，固定样品在未处理的细胞中表现出略高的背景，在BLM处理的细胞中评分为衰老的细胞百分比较高。在过夜储存的固定样品中也观察到了这种效应，并在四摄氏度下储存一周。显示了流式细胞术分选和通过形态和增殖标志物对富集衰老细胞群的验证。分选的衰老细胞显示出扩大的形态，正如预期的那样，通过用荧光鬼笔环肽染色肌动蛋白来可视化。通过免疫荧光染色也观察到增殖标志物Ki67信号的降低。最后，评估了用化疗药物治疗的肿瘤衰老的定量。组织中的 X-gal 染色相对较弱，但在用阿霉素或聚乙二醇化脂质体阿霉素治疗的肿瘤中，蓝色染色很明显，特别是在通过 DDAO-半乳糖苷流动测定也获得衰老阳性的肿瘤中。正如预期的那样，仅盐水肿瘤表现出可以忽略不计的衰老。在这里，与任何活细胞测定一样，方案步骤应有效但温和地执行。应避免长时间的染色程序或超过上述时间的孵育。如果对衰老细胞进行流式细胞术分选，则可以将它们放回培养物中进行免疫测定，或裂解并进行处理以进行组学分析，包括转录组学或蛋白质组学。这项技术使我们的实验室和其他人能够识别衰老肿瘤细胞的新特征，包括DNA损伤，蛋白质表达和代谢变化的定量。

Research

JoVE Journal

Cancer Research

Far-Red Fluorescent Senescence-Associated &#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

流动 Cytometry-based 药物筛选系统对促进细胞分化胶质母细胞瘤小分子的鉴定

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

科研

癌症研究

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

单细胞下游特征的靶细胞预富集与全基因组扩增

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

流式细胞术对小鼠原发性胰腺导管腺癌原位同种异体 (自体) 免疫分型的研究

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

肿瘤免疫和非免疫微环境在小鼠皮下肿瘤中的富集与鉴定

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

磷流式细胞仪与荧光细胞条形码用于单细胞信号分析和生物标志物发现

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Detection of Protease Activity by Fluorescent Peptide Zymography

荧光肽酶谱法检测蛋白酶活性

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

Cell Cycle-specific Measurement of ÃƒÅ½Ã‚Â³H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

细胞周期特异性测量βH2AX和细胞凋亡后热源毒性应力通过流细胞测定

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

用于低肿瘤含量组织中肿瘤富集的自动解剖方案

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly ...