Name: a flow cytometry-based cell surface protein binding assay for assessing selectivity and specificity of an anticancer aptamer
Uploaded: 2022-09-13T14:00:00
Description: Watch this Scientific Journal Video about A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer at JoVE.com

作者感谢心理和身体健康与临床翻译研究所 （IMPACT） SEED 资金、迪肯大学的“阿尔弗雷德·迪肯博士后研究奖学金”计划和“澳大利亚政府研究培训计划奖学金”。

注意：在开始实验之前，请穿戴个人防护设备，包括实验室外套，手套和护目镜。有关本协议中使用的材料、试剂、设备和软件的详细信息，请参阅 材料表 。
1. 测定所需的缓冲液
<ol><li>在实验当天新鲜制备本实验所需的缓冲液 - 适配体折叠所需的SELEX缓冲液，封闭缓冲液（BB），洗涤缓冲液（WB）和结合缓冲液（BiB）（表1），并将它们...

<ol>
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M., Shigdar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triple-negative+breast+cancer+brain+metastasis:+an+update+on+druggable+targets,+current+clinical+trials,+and+future+treatment+options.">Triple-negative breast cancer brain metastasis: an update on druggable targets, current clinical trials, and future treatment options.</a> Drug Discovery Today. , (2022).</li><li>Macdonald, 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=Development+of+a+bifunctional+aptamer+targeting+the+transferrin+receptor+and+epithelial+cell+adhesion+molecule+(EpCAM)+for+the+treatment+of+brain+cancer+metastases.">Development of a bifunctional aptamer targeting the transferrin receptor and epithelial cell adhesion molecule (EpCAM) for the treatment of brain cancer metastases.</a> ACS Chemical Neuroscience. 8 (4), 777-784 (2017).</li><li>Macdonald, 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=Bifunctional+aptamer-doxorubicin+conjugate+crosses+the+blood-brain+barrier+and+selectively+delivers+its+payload+to+EpCAM-positive+tumor+cells.">Bifunctional aptamer-doxorubicin conjugate crosses the blood-brain barrier and selectively delivers its payload to EpCAM-positive tumor cells.</a> Nucleic Acid Therapeutics. 30 (2), 117-128 (2020).</li><li>Shigdar, S., Agnello, L., Fedele, M., Camorani, S., Cerchia, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+cancer+cells+by+cell-SELEX:+use+of+aptamers+for+discovery+of+actionable+biomarkers+and+therapeutic+applications+thereof.">Profiling cancer cells by cell-SELEX: use of aptamers for discovery of actionable biomarkers and therapeutic applications thereof.</a> Pharmaceutics. 14 (1), 28 (2021).</li><li>Rahimizadeh, K., 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=Development+of+cell-specific+aptamers:+recent+advances+and+insight+into+the+selection+procedures.">Development of cell-specific aptamers: recent advances and insight into the selection procedures.</a> Molecules. 22 (12), 2070 (2017).</li><li>Chen, 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=Development+of+cell-SELEX+technology+and+its+application+in+cancer+diagnosis+and+therapy.">Development of cell-SELEX technology and its application in cancer diagnosis and therapy.</a> International Journal of Molecular Sciences. 17 (12), 2079 (2016).</li><li>Shigdar, 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=The+use+of+sensitive+chemical+antibodies+for+diagnosis:+detection+of+low+levels+of+EpCAM+in+breast+cancer.">The use of sensitive chemical antibodies for diagnosis: detection of low levels of EpCAM in breast cancer.</a> PLoS One. 8 (2), 57613 (2013).</li><li>Ni, 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=Role+of+the+EpCAM+(CD326)+in+prostate+cancer+metastasis+and+progression.">Role of the EpCAM (CD326) in prostate cancer metastasis and progression.</a> Cancer and Metastasis Reviews. 31 (3), 779-791 (2012).</li><li>Ni, 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=Epithelial+cell+adhesion+molecule+(EpCAM)+is+associated+with+prostate+cancer+metastasis+and+chemo/radioresistance+via+the+PI3K/Akt/mTOR+signaling+pathway.">Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer metastasis and chemo/radioresistance via the PI3K/Akt/mTOR signaling pathway.</a> The International Journal of Biochemistry &amp; Cell Biology. 45 (12), 2736-2748 (2013).</li><li>McKeague, M., Kruse, P. F., Patterson, M. K., 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> Tissue Culture. , 395-397 (1973).</li><li>McKeague, 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=The+minimum+aptamer+publication+standards+(MAPS+guidelines)+for+de+novo+aptamer+selection.">The minimum aptamer publication standards (MAPS guidelines) for de novo aptamer selection.</a> Aptamers. 6, 10-18 (2022).</li><li>Schoofs, G., Van Hout, A., D'huys, T., Schols, D., Van Loy, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flow+cytometry-based+assay+to+identify+compounds+that+disrupt+binding+of+fluorescently-labeled+CXC+Chemokine+ligand+12+to+CXC+Chemokine+receptor+4.">A flow cytometry-based assay to identify compounds that disrupt binding of fluorescently-labeled CXC Chemokine ligand 12 to CXC Chemokine receptor 4.</a> Journal of Visualized Experiments. (133), e57271 (2018).</li><li>McKinnon, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+an+overview.">Flow cytometry: an overview.</a> Current Protocols in Immunology. 120 (1), 1-11 (2018).</li><li>Kamiloglu, S., Sari, G., Ozdal, T., Capanoglu, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+cell+viability+assays.">Guidelines for cell viability assays.</a> Food Frontiers. 1 (3), 332-349 (2020).</li><li>Ruscito, A., DeRosa, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+binding+aptamers:+Selection+strategies,+characterization,+and+applications.">Small-molecule binding aptamers: Selection strategies, characterization, and applications.</a> Frontiers in Chemistry. 4, 14 (2016).</li><li>McKeague, 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=Comprehensive+analytical+comparison+of+strategies+used+for+small+molecule+aptamer+evaluation.">Comprehensive analytical comparison of strategies used for small molecule aptamer evaluation.</a> Analytical Chemistry. 87 (17), 8608-8612 (2015).</li><li>Henri, J., Bayat, N., Macdonald, J., Shigdar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+using+nucleic+acid+aptamers+in+cell+based+assays.">A guide to using nucleic acid aptamers in cell based assays.</a> Aptamers. 23, (2019).</li><li>Mao, 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=The+mechanism+and+regularity+of+quenching+the+effect+of+bases+on+fluorophores:+the+base-quenched+probe+method.">The mechanism and regularity of quenching the effect of bases on fluorophores: the base-quenched probe method.</a> Analyst. 143 (14), 3292-3301 (2018).</li><li>McKeague, 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=Analysis+of+in+vitro+aptamer+selection+parameters.">Analysis of in vitro aptamer selection parameters.</a> Journal of Molecular Evolution. 81 (5), 150-161 (2015).</li><li>Chen, B., 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+negative+surface+charges+of+cancer+cells+by+multifunctional+nanoprobes.">Targeting negative surface charges of cancer cells by multifunctional nanoprobes.</a> Theranostics. 6 (11), 1887 (2016).</li><li>Shigdar, 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=RNA+aptamers+targeting+cancer+stem+cell+marker+CD133.">RNA aptamers targeting cancer stem cell marker CD133.</a> Cancer Letters. 330 (1), 84-95 (2013).</li><li>Amraee, M., Oloomi, M., Yavari, A., Bouzari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+aptamer+identification+and+characterization+for+E.+coli+O157+detection+using+cell+based+SELEX+method.">DNA aptamer identification and characterization for E. coli O157 detection using cell based SELEX method.</a> Analytical Biochemistry. 536, 36-44 (2017).</li><li>Yu, X. -. 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开发新适配体的主要挑战是缺乏适用于该过程不同步骤的标准指南。McKeague等人最近证明了一些相关的挑战，这些挑战导致出版物中数据的呈现不明确，并且无法复制该研究。他们提出了表征适配体所必需的基本准则19。核酸适配体结合测定是筛选和/或表征核酸适配体23的关键步骤，该试剂被该领域的研究人员广泛使用。由于没有单一的指南来显示分步方案，因...

癌症仍然是全球死亡的主要原因之一1。尽管近几十年来癌症治疗有了显着改善，但抗癌药物的开发仍然是一个备受争议的话题。这是因为化疗作为癌症治疗的支柱，伴随着严重的副作用，限制了患者对治疗的依从性。此外，化疗引起的癌症对治疗的耐药性限制了其作为医疗干预的唯一选择的应用。单克隆抗体（mAb）的应用增强了对癌症治疗的反应2。使用mAb的基本原理是提高化疗药物的疗效并尽量减少其不良反应。然而，单克隆抗体的管理也成为一项挑战。这不仅是因为mAb诱导的免疫反应，还因为动物依赖性和昂贵的生产成本以及困难的储存条件3。1990年代4 中核酸适配体的引入为癌症治疗带来了新的希望，因为核酸适配体的应用可以解决与mAb相关的挑战。
核酸适配体是专门为特定靶标产生的短核酸序列。通过指数富集（SELEX）系统进化配体是适配体生产的常用方法。在SELEX中，目标蛋白与随机核苷酸序列文库一起孵育，并通过一系列迭代循环，纯化该蛋白特异性适配体。核酸适配子与mAb具有相似的靶标选择性和特异性，因此该领域的药物开发显示出广阔的未来应用前景。癌症生物标志物特异性适配体可用作单一药物和癌症诊断工具5，6，7。由于其纳米尺寸的结构，这些适配体还可以作为药物载体，将细胞毒性剂特异性地输送到肿瘤8。这将提高靶向药物递送的疗效，并减少化疗相关的脱靶不良反应。此外，这些纳米药物具有高组织渗透率，这使它们成为深部肿瘤药物递送和治疗的理想候选者。核酸适配体还可以设计成靶向在血脑屏障（BBB）上表达的转运蛋白，以改善药物向脑肿瘤的输送9。这种适配体的一个很好的例子是双功能适配体，靶向转铁蛋白受体（TfR）10以增强跨BBB的药物递送，并将细胞毒性药物有效载荷递送到肿瘤细胞11。
尽管核酸适配体具有所有优点，但该领域的药物开发尚未产生上市的、成功的抗癌药物。其中一个原因可能是缺乏该领域研究人员可以在全球范围内遵循的标准和可重复的方法。在本文中，展示了适配体与细胞表面表达的天然蛋白质结合的分步方案。该方案是抗癌适配体临床前评估的先决条件步骤。进行该测定以显示从SELEX或已发布的适配体序列中收集的纯化适配体的选择性和特异性，以确认选择性和特异性。这种基于流式细胞术的测定是一种快速、可靠、灵敏的测定，可准确显示适配体的选择性和特异性，其中核酸适配体正在针对细胞表面的蛋白质进行测试12，13，14。该方法使用本文15所示的EpCAM特异性适配体的结合进行了演示。EpCAM作为一种跨膜糖蛋白，在肿瘤细胞信号传导、进展、迁移和转移中发挥作用16，17。为了显示该适配体的选择性和特异性，使用了EpCAM阳性和阴性癌细胞。先前开发的EpCAM特异性适配体TEPP（5′-GC GCG GTAC CGC GC TA ACG GA GGTTGCG TCC GT-3′）和阴性对照适体TENN（5′-GC GCG TGCA CGC GC TA ACG GA TTCCTTT TCC GT-3）分别用作EpCAM结合和非结合适配体10。TEPP和TENN的3'端都用TYE665荧光团标记。
TEPP是一种双功能适配体，一端靶向EpCAM，另一端靶向TfR。这使得TEPP成为EpCAM+ 脑肿瘤药物递送的合适候选者。使用其TfR特异性末端，TEPP穿过血脑屏障，并使用EpCAM特异性末端找到肿瘤并将其货物（例如，细胞毒性药物）输送到肿瘤。TENN具有与TEPP相似的长度和2D结构，但它对EpCAM或TfR没有亲和力，因此是一种合适的阴性对照适配体。本文使用TEPP和TENN，使用流式细胞术测试适配体与靶蛋白的结合。该协议适用于细胞特异性适配体的开发。它也适用于文献中可用的适配体序列的进一步补充和确认分析。该协议也可以由那些刚进入适配体领域的人使用，他们正在考虑将以前发表的适配体用于他们的研发（R&D）目的。本文研究了文献中可用的两种适配体序列。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tubes with attached lid</td>
			<td>Sigma-Aldrich</td>
			<td>T6649</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL CellStar blue screw cap, conical bottom tube</td>
			<td>Greiner Bio One</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL serological pipettes</td>
			<td>Greiner Bio One</td>
			<td>606180</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSCanto II Flow Becton Dickinson Cytometer</td>
			<td>Becton Dickinson</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva V9.0</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA), Lyophilized powder</td>
			<td>Sigma-AldrichTM</td>
			<td>A7906-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-line Hemocytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM) High Glucose Media Powder</td>
			<td>Life Technologies</td>
			<td>12100046</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate- Buffered Saline (DPBS)</td>
			<td>Life Technologies</td>
			<td>21300025</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo, LLC 10.8.1</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum (FBS)</td>
			<td>Bovogen</td>
			<td>SFBS-F</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T</td>
			<td>American Type Culture Collection</td>
			<td>ACS-4500</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell 150i CO2 Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Heraeus Megafuge 16R Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>American Type Culture Collection</td>
			<td>CRM-HTB-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate, PS, 96 well, F-bottom (Chimney well), Black</td>
			<td>Greiner Bio One</td>
			<td>655076</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniAmp Thermal Cycler</td>
			<td>Thermo Fisher Scientific</td>
			<td>A37834</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS) tablets</td>
			<td>Life Technologies</td>
			<td>18912014</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrogen- and RNase-free ultrapure water</td>
			<td>Milli-Q</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T75 Cell Culture flask</td>
			<td>Cellstar</td>
			<td>658170</td>
			<td></td>
		</tr>
		<tr>
			<td>TENN</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A</td>
			<td>5&#8242;-GC GCG TGCA CGC GC TA ACG GA TTCCTTT TCC GT-3</td>
		</tr>
		<tr>
			<td>TEPP</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A</td>
			<td>5&#8242;-GC GCG GTAC CGC GC TA ACG GA GGTTGCG TCC GT-3&#8242;</td>
		</tr>
		<tr>
			<td>Transfer RNA (tRNA)</td>
			<td>Sigma-Aldrich</td>
			<td>R8508-5X1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Life Technologies</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>15400054</td>
			<td></td>
		</tr>
	</tbody>
</table>

a flow cytometry-based cell surface protein binding assay for assessing selectivity and specificity of an anticancer aptamer

开发抗癌适配体的一个关键挑战是有效地确定开发的适配体对目标蛋白的选择性和特异性。由于其与单克隆抗体相比的几个优点，适配体的开发在癌症研究人员中获得了极大的欢迎。通过指数富集（SELEX）对配体进行系统进化是开发目标蛋白质特异性适配体的最常见方法。在SELEX之后，快速有效的结合测定加快了鉴定过程，确认了适配体的选择性和特异性。 
本文解释了一种基于流式细胞术的上皮细胞粘附分子特异性适配体（EpCAM）的分步流式细胞术结合测定。跨膜糖蛋白EpCAM在大多数癌中过表达，并在癌症的发生、进展和转移中发挥作用。因此，它是靶向药物递送到肿瘤的有价值的候选者。为了评估适配体对膜结合EpCAM的选择性和特异性，需要EpCAM阳性和阴性细胞。此外，还需要具有与EpCAM结合适配体相似的长度和二维（2D）结构的非结合性EpCAM适配体。结合测定包括不同的缓冲液（封闭缓冲液、洗涤缓冲液、孵育缓冲液和FACS缓冲液）和孵育步骤。 
核酸适配体与细胞系一起孵育。在孵育和洗涤步骤之后，将使用灵敏的流式细胞术测定法评估细胞。结果分析显示EpCAM特异性适配体与EpCAM阳性细胞结合，而不是EpCAM阴性细胞。在EpCAM阳性细胞中，与非结合适配体对照相比，这被描述为EpCAM适配体向右结合的带移。在EpCAM阴性细胞中，EpCAM结合和非结合适配体的相应条带重叠。这证明了EpCAM适配体的选择性和特异性。虽然该协议侧重于EpCAM适配体，但该协议适用于其他已发布的适配体。

新药发现和开发的一个重要方面是确保候选药物的选择性和特异性。这意味着候选药物应该能够区分不同的细胞，并且只影响感兴趣的细胞群（选择性）。使用在目标蛋白表达方面不同的细胞系研究选择性。在这项研究中，MDA-MB-231和HEK 293T细胞系被选为EpCAM阳性和阴性细胞。特异性是另一个决定因素，表明感兴趣的蛋白质仅对单个候选药物有反应。在这里，通过使用EpCAM非结合适体TENN，表明只有TE...

Watch this Scientific Journal Video about A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer at JoVE.com

A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer

抗癌适配体开发的必要步骤是测试其与靶标的结合。我们展示了一种基于流式细胞术的测定来研究这种结合，强调了包括阴性对照适配体和对该特定蛋白质呈阳性或阴性的癌细胞的重要性。

一种基于流式细胞术的细胞表面蛋白结合测定，用于评估抗癌适配体的选择性和特异性

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target ...

该协议将帮助刚接触适配体的研究人员能够在他们的项目中使用它们，并了解完成协议所需的各个小步骤。核酸适配体用途广泛，该协议证明了它们在免疫表型测定中的简单性和简单性。适配体可用于诊断和治疗应用。该协议是为确认其特异性和敏感性而采取的第一步。虽然这种特殊的测定表明了一个细胞表面受体的染色，但核酸适配体可以很容易地用于多参数流式细胞术，提供高水平的表型知识。使用核酸适配体的重要方面是确保它们在使用前正确折叠，以及缓冲液和离子强度条件与选择时相同。收集并丢弃烧瓶中的培养基后，加入三毫升PBS并将其铺在细胞上。向每个烧瓶中加入一毫升0.25%的胰蛋白酶-EDTA后，在37摄氏度下孵育5至10分钟，并在显微镜下观察细胞的分离。接下来，向细胞中加入一毫升完全培养基，并上下移液细胞以制成单细胞悬液。将细胞移液到适当的管中，并以200 G离心五分钟。弃去上清液后，将细胞重悬于一毫升新鲜培养基中，并通过用台盼蓝稀释一定体积的细胞悬液，使用台盼蓝染色来计数细胞。在血细胞计数器和盖玻片之间分配约15微升混合物以计数细胞。收集所需数量的细胞，确保每个测试样品有十万个细胞。然后将细胞在37摄氏度下孵育两个小时，以使酶分离后细胞膜上感兴趣的蛋白质稳定。孵育两个小时后，将细胞以500 G离心五分钟。弃去上清液后，将细胞重悬于500微升封闭缓冲液中。将细胞在 4 摄氏度下孵育 30 分钟，间歇混合。在这30分钟的孵育过程中，通过制备手稿中描述的两倍浓度的适配体来进行适配体折叠。然后根据所需的折叠条件将核酸适配体与热循环仪混合并孵育，同时确保保护核酸适配体免受光照。孵育30分钟后，如前所述离心细胞并除去上清液。然后加入一毫升洗涤缓冲液并再次离心细胞。除去上清液后，将细胞重悬于适当体积的结合缓冲液中。接下来，将50微升重悬的细胞移液到冰冷的96孔黑板的每个孔中。然后将50微升适配体移液到50微升体积的细胞上，混合并在4摄氏度的黑暗中孵育30分钟。离心并除去上清液后，小心地将沉淀重悬于洗涤缓冲液中，然后再次以500G离心五分钟。然后重悬于100微升洗涤缓冲液中进行流式细胞术分析，同时重复洗涤步骤两次。首先，打开流式细胞仪，然后打开计算机。接下来，打开流式细胞术分析软件，登录程序，然后在细胞仪选项卡下运行流体启动。若要创建新实验，请在“实验”选项卡下，单击“新建文件夹”，然后将文件夹命名为斜杠实验。单击新文件夹以突出显示它。然后再次在实验标签下，点击新建实验并相应地命名实验。要添加第一个示例斜杠样本，请在试验选项卡下单击新样本并相应地命名样本。要添加试管样品，请突出显示该试样，然后在实验选项卡下单击新建试管。然后添加适当数量的管子和名称。若要准备所需的图形，请在工作表选项卡下打开一个新工作表。弹出新的工作表窗口后，准备侧向散射与前向散射的点印迹图以选择感兴趣的总体。准备前向散射高度与前向散射区域的点印迹图，以选择单细胞群。接下来，准备针对目标荧光团的事件数的直方图。在开始流式细胞术之前，确保用于控制样品采集的采集仪表板、用于调整电压参数的检测器和细胞仪以及包含所有图表的工作表处于打开状态。首先，运行未处理的细胞。确保指向管子的箭头是绿色的。如果此箭头不是绿色的，请单击箭头使其变为绿色。使用移液器，将每个样品从96孔黑板转移到流式细胞术管中。在采集仪表板上，选择要记录的适当数量的事件，将流速更改为低，然后单击采集数据。调整FSC和SCC参数的电压，确保细胞群集中在点图中，并且没有细胞接触图形的任一轴，以避免丢失感兴趣的细胞。通过在前向和侧向散射密度图中识别和选择感兴趣的种群来定义第一个门，同时排除碎片，碎片构成了具有最低前向散射信号的种群。通过排除双峰细胞群来定义第二个门，因为双峰细胞会显着影响结果和结论。将采集速度提高到中或高以更快地分析样品，但不要超过每秒 200 到 300 个事件。然后单击“记录数据”。调整电压和门控并记录数据后，取出样品并单击下一个管。插入下一个样品并重复所有对照和测试样品的记录数据。收集完所有数据后，通过运行三管 50% 漂白剂、FACSRinse 和超纯水来洗涤流式细胞仪，每管以高流速洗涤 5 分钟。然后从细胞仪下拉菜单中，单击流控系统关闭。将结果作为 fsc 文件导出到 USB 驱动器进行传输。使用分析软件分析它们，方法是按新建按钮创建一个新文档和窗口来处理分析，然后将示例文件拖到新窗口中。双击以打开未染色的样品。要门控单细胞群，请选择 P1 群体并双击 P1 群体以创建 FSCH 与 FSCA 图。双击门控单细胞以针对使用的荧光染料创建事件的直方图。在原始窗口中，选择 P1 和单个单元格并将它们拖到所有样本中，以便所有样本现在都包含相同的门控。接下来，单击布局编辑器按钮打开布局窗口，然后将两个样本相互拖动以创建叠加直方图。在EpCAM阳性MDA-MB-231细胞中，叠加代表用TEPP和TENN处理的细胞的直方图显示TEPP处理的细胞向右移动，而TENN处理的细胞表明TEPP适配体与目标蛋白质的结合发生。在阴性对照中，覆盖TEPP和TENN直方图的HEK 293T细胞没有反映任何偏移，表明在表达EpCAM的细胞中，TEPP是附着在其受体上的EpCAM适配体。此外，在EpCAM阴性细胞中未观察到结合，这证实了开发的适体的选择性和特异性。确保核酸适配体避光，将核酸适配体和细胞混合在平板上，并在流式细胞仪上设置正确的电压。这在很大程度上取决于核酸适配体的未来应用。例如，我们专注于药物输送。因此，我们的下一步将是使用荧光显微镜评估适配体是否内化。该协议已经证明了核酸适配体在免疫表型测定中取代单克隆和多克隆抗体是多么容易。

Research

JoVE Journal

Cancer Research

A Method of Targeted Cell Isolation via Glass Surface Functionalization

通过玻璃表面功能靶细胞提取方法

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual ...

科研

癌症研究

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

胰腺癌的荧光原位小鼠模型

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

光动力疗法的抗癌功效肺癌靶向纳米粒子

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

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 ...

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

二维凝胶电泳结合质谱法分析人垂体腺瘤组织蛋白质组

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

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 ...

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 ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

使用自动活单元成像仪的简单迁移/入侵工作流

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

评估癌细胞3D球形培养中的细胞活力和死亡

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...