We thank Hannah Puzas for assistance with system design and configuration, and Kevin Khovananth for technical advice. Funding for this work was provided by The Hearst Foundations and the Providence Portland Medical Foundation.

<ol>
	<li>Maecker, H. T., McCoy, J. P., Nussenblatt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardizing+immunophenotyping+for+the+Human+Immunology+Project.">Standardizing immunophenotyping for the Human Immunology Project.</a> Nat. Rev. Immunol. 12 (3), 191-200 (2012).</li><li>Craig, F. E., Foon, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+immunophenotyping+for+hematologic+neoplasms.">Flow cytometric immunophenotyping for hematologic neoplasms.</a> Blood. 111 (8), 3941-3967 (2008).</li><li>Johansson, U., Bloxham, D., 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=Guidelines+on+the+use+of+multicolour+flow+cytometry+in+the+diagnosis+of+haematological+neoplasms.">Guidelines on the use of multicolour flow cytometry in the diagnosis of haematological neoplasms.</a> Br. J. Haematol. 165 (4), 455-488 (2014).</li><li>Schnizlein-Bick, C. T., Spritzler, 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=Evaluation+of+TruCount+Absolute-Count+Tubes+for+Determining+CD4+and+CD8+Cell+Numbers+in+Human+Immunodeficiency+Virus-Positive+Adults.">Evaluation of TruCount Absolute-Count Tubes for Determining CD4 and CD8 Cell Numbers in Human Immunodeficiency Virus-Positive Adults.</a> Clin. Vaccine Immunol. 7 (3), 336-343 (2000).</li><li>Cossarizza, A., De Biasi, 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=Cytometry,+immunology,+and+HIV+infection:+Three+decades+of+strong+interactions.">Cytometry, immunology, and HIV infection: Three decades of strong interactions.</a> Cytometry A. 83 (8), 680-691 (2013).</li><li>Hensley, T. R., Easter, A. 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=Enumeration+of+Major+Peripheral+Blood+Leukocyte+Populations+for+Multicenter+Clinical+Trials+Using+a+Whole+Blood+Phenotyping+Assay.">Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay.</a> J. Vis. Exp. (67), e4302 (2012).</li><li>Streitz, M., Miloud, T., 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=Standardization+of+whole+blood+immune+phenotype+monitoring+for+clinical+trials:+panels+and+methods+from+the+ONE+study.">Standardization of whole blood immune phenotype monitoring for clinical trials: panels and methods from the ONE study.</a> Transplant. Res. 2 (1), 17 (2013).</li><li>Hensley-McBain, T., Heit, A., De Rosa, S. C., McElrath, M. J., Andersen-Nissen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+whole+blood+phenotyping+assay+for+enumeration+of+peripheral+blood+leukocyte+populations+in+multicenter+clinical+trials.">Optimization of a whole blood phenotyping assay for enumeration of peripheral blood leukocyte populations in multicenter clinical trials.</a> J. Immunol. Methods. 411, 23-36 (2014).</li><li>Green, C. L., Brown, L., Stewart, J. J., Xu, Y., Litwin, V., Closkey, T. W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+the+validation+of+flow+cytometric+testing+during+drug+development:+I+instrumentation.">Recommendations for the validation of flow cytometric testing during drug development: I instrumentation.</a> J. Immunol. Methods. 363 (2), 104-119 (2011).</li><li>O'Hara, D. M., Xu, Y., Liang, Z., Reddy, M. P., Wu, D. Y., Litwin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+the+validation+of+flow+cytometric+testing+during+drug+development:+II+assays.">Recommendations for the validation of flow cytometric testing during drug development: II assays.</a> J. Immunol. Methods. 363 (2), 120-134 (2011).</li><li>Owens, M. A., Vall, H. G., Hurley, A. A., Wormsley, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+and+quality+control+of+immunophenotyping+in+clinical+flow+cytometry.">Validation and quality control of immunophenotyping in clinical flow cytometry.</a> J. Immunol. Methods. 243 (1-2), 33-50 (2000).</li><li>Kalina, T., Flores-Montero, 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=EuroFlow+standardization+of+flow+cytometer+instrument+settings+and+immunophenotyping+protocols.">EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols.</a> Leukemia. 26 (9), 1986-2010 (2012).</li><li>Calvelli, T., Denny, T. N., Paxton, H., Gelman, R., Kagan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guideline+for+flow+cytometric+immunophenotyping:+a+report+from+the+National+Institute+of+Allergy+and+Infectious+Diseases,+Division+of+AIDS.">Guideline for flow cytometric immunophenotyping: a report from the National Institute of Allergy and Infectious Diseases, Division of AIDS.</a> Cytometry A. 14 (7), 702-715 (1993).</li><li>Biancotto, A., McCoy, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+the+human+immunome:+the+complexity+of+comprehensive+leukocyte+immunophenotyping.">Studying the human immunome: the complexity of comprehensive leukocyte immunophenotyping.</a> Curr. Top. Microbiol. Immunol. 377, 23-60 (2014).</li><li>Perfetto, S. P., Ambrozak, D., Nguyen, R., Chattopadhyay, P., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+assurance+for+polychromatic+flow+cytometry.">Quality assurance for polychromatic flow cytometry.</a> Nat. Protoc. 1 (3), 1522-1530 (2006).</li><li>Yong, P. F. K., Salzer, U., Grimbacher, B. <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+costimulation+in+antibody+deficiencies:+ICOS+and+common+variable+immunodeficiency.">The role of costimulation in antibody deficiencies: ICOS and common variable immunodeficiency.</a> Immunol. Rev. 229 (1), 101-113 (2009).</li><li>Donnenberg, A. D., Donnenberg, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rare-Event+Analysis+in+Flow+Cytometry.">Rare-Event Analysis in Flow Cytometry.</a> Clin. Lab. Med. 27 (3), 627-652 (2007).</li><li>Biancotto, A., Fuchs, J. C., Williams, A., Dagur, P. K., McCoy, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+dimensional+flow+cytometry+for+comprehensive+leukocyte+immunophenotyping+(CLIP)+in+translational+research.">High dimensional flow cytometry for comprehensive leukocyte immunophenotyping (CLIP) in translational research.</a> J. Immunol. Methods. 363 (2), 245-261 (2011).</li><li>Villanova, F., Di Meglio, 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=Integration+of+Lyoplate+Based+Flow+Cytometry+and+Computational+Analysis+for+Standardized+Immunological+Biomarker+Discovery.">Integration of Lyoplate Based Flow Cytometry and Computational Analysis for Standardized Immunological Biomarker Discovery.</a> PloS One. 8 (7), e65485 (2013).</li><li>BD biosciences. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optimizing Experiments Using BD Horizon Brilliant Polymer Conjugates. , (2014).</li><li>Malergue, F., van Agthoven, A., Scifo, C., Egan, D., Strous, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automation+of+a+Phospho-STAT5+Staining+Procedure+for+Flow+Cytometry+for+Application+in+Drug+Discovery.">Automation of a Phospho-STAT5 Staining Procedure for Flow Cytometry for Application in Drug Discovery.</a> J. Biomol. Screen. 20 (3), 416-421 (2015).</li><li>Hasan, M., Beitz, 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=Semi-automated+and+standardized+cytometric+procedures+for+multi-panel+and+multi-parametric+wholeblood+immunophenotyping.">Semi-automated and standardized cytometric procedures for multi-panel and multi-parametric wholeblood immunophenotyping.</a> Clin. Immunol. 157 (2), 261-276 (2015).</li><li>Maecker, H. T., McCoy, 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+model+for+harmonizing+flow+cytometry+in+clinical+trials.">A model for harmonizing flow cytometry in clinical trials.</a> Nat. immunol. 11 (11), 975-978 (2010).</li></ol>

YK，ILG，TLM，WLM，DPH和KSB:没有竞争的经济利益。 ANT是Beckman Coulter公司的员工，这个视频文章的出版公司是由Beckman Coulter，Inc赞助

外周血免疫是获得分析上市个别响应的免疫治疗至关重要。挑战在于标准化检测控制实验对实验变异1,14。变异性的一个主要来源在于样品的人的操纵。因此，可以想到，样本处理的部分或全部自动化将促进实验对实验变异性-1,14-显着减少。在这个协议中，我们报道了成功的努力通过导入装有二维条码阅读器，用于在全血免疫样品染色的自动化液体处理器，以尽量减少测定变异性。 在设计中，我们的方法是通用的，可以通过添加额外的"基地鸡尾酒"来定义他们监视其他免疫子集。这样的子集包括T辅助细胞，调节性T细胞，B细胞，NK细胞，树突细胞，和单核细胞（手稿中准备）18。我们通过引入额外的1标记适应财团的建议抗体面板。细胞群用"基地鸡尾酒"共轭荧光染料以最小排放到明亮的紫罗兰色421（BV421），藻红蛋白（PE）标识，别藻蓝蛋白（APC）的探测器，因为我们想保留这些渠道的检测诱导抗原。此功能不仅保证了最高灵敏度监测免疫细胞的激活状态，但也使我们在这三个荧光最常与抗诱导标志物的抗体结合的新的激活标记灵活的住宿。因此，我们的方法不仅适用于全血免疫鸡尾酒制剂，但也用于染色其它样品，包括从分列组织（例如，肿瘤）中回收外周血单核细胞和细胞是有用的。 成功的前奏我们的方法元币种需要实验室器皿准备特别注意步骤，编程和该方法的执行。二维条码管制备包括具有人可读的标签和抗体的转移到指定的管标签。为了防止错误的引入，我们建议两个人这样做。只要更改了电子表格，调查人员应验证该方法是否正常。编程过程中选择合适的移液的方法，确保成功的液体转移。 LLS使移液未经抗体管，为此，我们请使用P50或P200导电尖端的底部得到沉淀杂物。 LLS可能不是P1000提示一个不错的选择为LLS是P1000的提示更敏感，有时错误地气泡存在触发。在实验室器具定义的高度应为每个仪器可能发生作为次优的液体传输来调整，例如，如果没有提示的边缘之间有足够的空间和管子的底部。 如图 12所示，如果"基地鸡尾酒"和/或"激活鸡尾酒"都没有很好涡旋，它可能会导致次优的染色。 我们想过用冻干试剂细胞染色作为一种替代方法，以减少与试剂分装19相关的变异。然而，灿烂紫色染料的聚合物缀合物是已知的相互引起的非特异性信号进行交互。这样，加在冻干试剂两个以上聚合物缀合物（例如，BV421和BV650 等）可能会引起非特异性的信号（例如，在BV421 +群体 BV650背景信号增加）20。此外，冻干试剂缺乏包括新染色的灵活性。他们通常比较昂贵，并且需要大量订购。由于这些原因，我们选择使用搭载二维条码管自动化液体处理程序。虽然ŧ碟刹时间来建立较难，导致了前期投资购买仪器，从长远来看，这些因素将会由生产力的提高和检测的重复性进行补偿。事实上，几组先前报告的自动液体处理器的成功整合到他们的免疫或类似的应用程序21,22的工作流程。对于流式细胞仪分析的自动化解决方案也可从商业来源（FACS SPA三，自动鸡尾酒制备工作站，和FlowStainer）。这进一步表明有用于免疫自动鸡尾酒制剂极其需要。 掌握这一技术后，我们设想，全自动全血免疫的发展将进一步降低实验对实验变异，可能使全血免疫即使是在多中心临床试验设置23可行的。用裂解是我们已经开始^ h助手自动裂解和洗涤步骤。我们还设想，抗体的二维条码管的体积和试剂分的跟踪自动化决心将大大提高分别抗体的库存和在我们的方法中的质量控制。

的人末梢血免疫确定在免疫细胞亚群1的定量和定性的改变。它提供了洞察和行动性，预测性的生物标志物的发现艾滋病的机制，并促进联合免疫疗法的发展。因此，免疫的验证和标准化是相当感兴趣的学术研究人员，临床实验室，和工业的领域。 虽然外周血通过流式细胞方法免疫目前用于血液恶性肿瘤2,3和人类免疫缺陷病毒（HIV）感染4,5，免疫治疗需求的外周血可靠免疫在验证和标准化，因为它具体考虑的临床管理要求对免疫细胞亚群和激活/抑制性受体1,6-8覆盖范围更加广泛。帅客ssful免疫需要仔细标准化，以减少相关的仪器设置和细胞染色过程9,10实验对实验的可变性。虽然流式细胞仪的仪器设置标准化是公认的解决前者关注9,11,12，它如何在不限制免疫细胞亚群和他们的激活状态的覆盖范围最小化与细胞染色变化仍不清楚。 作为待检测的抗原的数目的增加，所以也没有用于误差和变异由于不理想的试剂分注和交叉污染的机会。成立于80年代末用于临床实验室检测为使用抗凝全血phenoptypic分析方法。这些相同的方法服务于科研实验室样品制备的需求。重要的是，在临床实验室中使用的抗体的鸡尾酒通常较不复杂和AVAilable预滴定和从制造商预混合。仅需要预混合抗体混合物的单个传输。在研究设置，10-16抗体的抗体鸡尾酒是典型的。每个鸡尾酒必须通过实验室验证的稳定性或制备的每个实验前新鲜。准备多种抗体鸡尾酒的样本可能带来50-80个人抗体的移液，既繁琐又容易出错的任务。 鸡尾酒制剂的自动化拥有手动鸡尾酒制剂几个优点，例如更少的错误，提高了移液的准确度，甚至可能降低试剂的浪费。这里，我们报告成功引入装有的二维（2D）条形码读取器到免疫的细胞染色过程中尽量减少样品制备相关的变异的自动液体处理器的。

<table border="0" cellpadding="0" cellspacing="0" width="1546">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:613px;">BD LSRFortessa</td>
			<td style="width:188px;">BD Biosciences</td>
			<td style="width:116px;"></td>
			<td style="width:629px;">Flow cytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NXp Span-8 Laboratory Automation Workstation&#160;</td>
			<td>Beckman Coulter</td>
			<td>A31839</td>
			<td>Laboratory Automation Workstation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insert, Tube, 11 mm, White, for 1.5 mL Microfuge Tubes (case of 25)</td>
			<td>Beckman Coulter</td>
			<td>373696</td>
			<td>Accessary for 24-Position Tube Rack to accommodate microfuge tubes.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LLS kit</td>
			<td>Beckman Coulter</td>
			<td>719262</td>
			<td>Attach bottom of the deck in Laboratory Automation Workstation to enable liquid level sensing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-Position Tube Rack</td>
			<td>Beckman Coulter</td>
			<td>373661</td>
			<td>Tube rack to hold microfuge tubes for antibody cocktails or blood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BIOHAZARD AUTOCLAVE BAGS 12x24 IN. RED</td>
			<td>LabMart</td>
			<td>M111416</td>
			<td>Disposable autoclave bags for the Laboratory Automation Workstation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VisionMate High Speed 2D Barcode Reader</td>
			<td>Thermo Scientific</td>
			<td>AB-1850</td>
			<td>2D barcode reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8-Channel Handheld Screw Cap Capper/Decapper</td>
			<td>Thermo Scientific</td>
			<td>4105MAT</td>
			<td>For de-capping and capping 2D barcode tubes&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>02-681-335</td>
			<td>For making cocktail in or supplying blood speciment to the Laboratory Automation Workstation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CoolRack XT CFT24</td>
			<td>biocision</td>
			<td>BCS-534</td>
			<td>To hold sample tubes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.0 mL Matrix ScrewTop Amber Tubes in Latch Racks</td>
			<td>Thermo Scientific</td>
			<td>3741AMB</td>
			<td>2D barcode tubes to store fluorescent dye-cojugated antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biomek Span-8 P1000 Tips, Conductive, Pre-sterile with Barrier, 1025 &#181;L</td>
			<td>Beckman Coulter</td>
			<td>987925</td>
			<td>Tips for Laboratory Automation Workstation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biomek Span-8 P50 Tips, Conductive, Pre-sterile with Barrier, 50 &#181;L (Case of 10 racks)</td>
			<td>Beckman Coulter</td>
			<td>B01091</td>
			<td>Tips for Laboratory Automation Workstation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biomek Span-8 P250 Tips, Conductive, Pre-sterile with Barrier (Case of 10 racks)</td>
			<td>Beckman Coulter</td>
			<td>394627</td>
			<td>Tips for Laboratory Automation Workstation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD FACS Lysing Solution 10X Concentrate</td>
			<td>BD Biosciences</td>
			<td>349202</td>
			<td>RBC lysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Round-Bottom Polystyrene Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-5</td>
			<td>Sample tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD45 FITC</td>
			<td>BD Biosciences</td>
			<td>347463</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD3 Alexa Fluor 700</td>
			<td>eBioscience</td>
			<td>56-0038-42</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD4 PerCPCy5.5</td>
			<td>BD Biosciences</td>
			<td>341654</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-TCRgd BV650</td>
			<td>BD Biosciences</td>
			<td>564156</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-TCRab BV786</td>
			<td>BD Biosciences</td>
			<td>563825</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD8 APC-H7</td>
			<td>BD Biosciences</td>
			<td>560179</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CCR7 PE-CF594</td>
			<td>BD Biosciences</td>
			<td>562381</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD45RA PE-Cy7</td>
			<td>BD Biosciences</td>
			<td>337167</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-HLA-DR BV421</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">562804</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD278(ICOS) PE</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">557802</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD38 APC</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">340439</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD279(PD1) BV421</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">562516</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD357(GITR) PE</td>
			<td>eBioscience</td>
			<td style="width:116px;">12-5875-42</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD137(41BB) APC</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">550890</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD69 BV421</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">562884</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD314(NKG2D) PE</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">557940</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD134(OX40) APC</td>
			<td style="width:188px;">BioLegend</td>
			<td>350008</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Vacutainer Plastic Blood Collection Tubes with Sodium Heparin: Conventional Stopper</td>
			<td>Fisher Scientific</td>
			<td>02-689-6</td>
			<td>For collecting blood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin, Bovine Serum, Fraction V, RIA and ELISA Grade</td>
			<td>EMD Millipore</td>
			<td>126593</td>
			<td>For flow wash buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Azide</td>
			<td>Sigma-Aldrich</td>
			<td>S8032</td>
			<td>For flow wash buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin, sodium salt</td>
			<td>Affimetrix</td>
			<td>16920</td>
			<td>For flow wash buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hank&#8217;s Balanced Salt Solution (HBSS), 1X, with Calcium, Magnesium, without Phenol Red</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30588.02</td>
			<td>For flow wash buffer</td>
		</tr>
	</tbody>
</table>

a semi-automated approach to preparing antibody cocktails for immunophenotypic analysis of human peripheral blood

外周血通过流式细胞免疫仪确定疾病和治疗过程中外周血白细胞的频率和激活状态的改变。它具有预测治疗功效，并确定新的治疗目标的潜力。全血染色使用未经处理的血液，最大限度地减少可样品制备过程中可能出现的文物。然而，全血染色还必须在新鲜采集的血液进行，以确保样品的完整性。此外，最好是在同一天准备抗体的鸡尾酒，以避免串联染料的潜在不稳定性，防止灿烂的紫色染料之间试剂反应。因此，全血染色需要仔细标准化来控制内和实验间的差异。 这里，我们报告装有的二维（2D）条形码读取器的自动液体处理器的调配成使得antibod的标准方法Ÿ鸡尾酒流式细胞仪。抗体被转移到设置在数据库中编译一个96孔格式及其内容的二维条形码的管中。然后，液体处理器可以通过在数据库中引用抗体名称查找源抗体的小瓶。我们的方法消除了繁琐的协调源抗体管的定位。它提供的多功能性允许用户容易地通过修改的数据库，而不在每个测定中软件的方法重写脚本更改任何数目的抗体分配工艺细节，如特异性抗体的使用，体积，和目的地。 概念实验的证明取得了突出的间和分子内精密度，由一个11色，17 - 抗体流式细胞仪检测的重复编写证明。这些方法增加的总吞吐量，流式细胞仪检测，并协助每天准备的详细苯氧所需的复杂抗体鸡尾酒新鲜收集抗凝外周血typic表征。

Watch this Scientific Journal Video about A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood at JoVE.com

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

半自动化的方法准备抗体鸡尾酒人外周血中免疫表型分析

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

a-semi-automated-approach-to-preparing-antibody-cocktails-for

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.7K 次观看.  Providence Cancer Center, Providence Portland Medical Center. Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

视频: A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

科研

免疫与感染

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

扩展人外周血γδT细胞使用Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

制定唾液抗体多重免疫测定来测量人体暴露于环境中的病原体

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

单核细胞单层试验中用于评估Fcγ受体介导的吞噬作用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

半自动化的方法准备抗体鸡尾酒人外周血中免疫表型分析

摘要

探索更多视频

此视频中的章节

人外周血NK细胞的扩展，纯化和功能评估

扩展人外周血γδT细胞使用Zoledronate

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

从外周血人类同种异体抗原特异性T细胞的生成

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

制定唾液抗体多重免疫测定来测量人体暴露于环境中的病原体

人全血中NK细胞杀伤活性的流式细胞仪分析

单核细胞单层试验中用于评估Fcγ受体介导的吞噬作用

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

全血流式细胞术分析人单核细胞亚群的表征