我们感谢他们协助发展这种方法，手稿和视频杰西卡·琼斯，埃里卡·克拉克，康斯坦茨Ducar，唐娜·史密斯，罗伊·刘易斯，百合Apedaile，乔安妮·威斯纳，德文·亚当斯，科里·麦克贝恩和斯蒂芬Voght。 这项工作是由比尔和梅林达·盖茨基金会CAVD授予38645（MJM）和美国国立卫生补助UM1 AI068618和U01 AI069481（MJM）的支持。 EA-N。由美国国立卫生研究院资助T32 AI007140支持。我们感谢詹姆斯·B·彭德尔顿慈善信托基金的慷慨设备捐赠。

注：为了保护从光的荧光标记的抗体在生物安全柜的灯不亮，执行所有步骤。 1。抗体染色面板准备<ol><li>于表1中可以发现的抗体染色面板。抗体浓度应与全血通过滴定定义，并使用相同的流式细胞仪，将用于获得染色表型样本的设备和程序。 </li><li>一旦确定适当的染色滴度，所有的抗体结合成一个单一的混合物，的锁帽筒。添加流洗涤缓冲液（2％热灭活的小牛血清的Dulbecco PBS），使总体积为100μl。缩放的混合物被染色的样本的数目。该混合物在4℃下，可以存储多达八个星期。 </li></ol> 2。染色法<ol><li>如果采集的血液是被用于其他目的，在广告CONDITION（供电状态）这个实验中，设定的等份，留出更多的时间敏感的程序进行，将剩下的血。的等分试样可以在室温下贮存长达4小时静脉穿刺后没有显着的细胞的损失。 </li><li>验证在Trucount管和标签的管，以确定被染色的样品的底部，有一个完整的珠粒料。 HVTN临床试验，实验室数据管理系统（前沿科学和技术研究基金会，纽约州Amherst）用于标记和跟踪染色样品。 </li><li>记录的所有试剂的批号和到期日期。记录的Trucount的管珠计数管包装袋上的制造商所提供的数，确保包装袋上的批号相符的批号管。 </li><li>使用反向移液管准确移液管100微升的全血到Trucount管，上面的金属固定器。避免弄脏血液向下侧的管。 </li><li&gt;使用到Trucount管经常（向前）移液技术，吸移管100微升的混合抗体染色面板（请参阅表1）。盖上管和旋涡在低速混合约15秒。目视检查管，以确保在胎圈的颗粒完全溶解。 </li><li>在黑暗中在室温下（15-30℃）15分钟，孵育Trucount管。 </li><li>如果有必要，10×FACS裂解液的等分试样稀释到1X使用DIH 2 O。添加900微升1×FACS裂解溶液的试管。 </li><li>盖上管盖和涡彻底在低速下混合约15秒。向下推盖牢固地插入锁定与实验室膜管和密封的位置上。 </li><li>管存放在-65°C至-95°C，直到样品是准备运送到中央分析实验室，或在内部进行分析。样品是稳定的在这个阶段，在至少四个星期。如果运输或化验IMMEdiately，该步骤可以被省略。 </li></ol> 3。航运 注：以下说明利用的绝缘运输系统的SAF-T-PAK，专门用于运输B类豁免生物活性物质，根据国际航空运输协会（IATA）的规定。如果分析的样品在相同的位置，作为染色发生，到第4条。 <ol><li>样品可立即发货染色后，一旦被冻结在-65°C到-95°C。各管缠绕在染色标本中完全箔和地点。将染色标本盒里面多袋防漏的吸收材料。 </li><li>特卫强袋和密封防漏胶袋，内容收入囊中的空气尽可能少。 </li><li>将样品包（二次包装内的标本）内的棕色盒子内。 </li><li>将内部的褐色B牛里面的泡沫塑料胸部，确保到缩进以防止移位。 </li><li>填充泡沫塑料胸部用干冰（约8公斤），将盖子盖紧，放在胸前。 </li><li>装运箱和船舶用胶带牢牢生物物质，B类（UN3373）用适当的干冰（UN1845）标记的;按照国际航空运输协会PI-650的说明。 </li><li>一旦收到，样本被存储在-65℃至-95℃，直到对它们进行分析。 </li></ol> 4。解冻和流式细胞仪分析<ol><li>从冷冻机中取出染色的样品的流式细胞仪上收集之前，在黑暗中在室温下解冻。如果在一个以上的样品收集数据，规范流程，所有的管子惊人的融化，使管不是停留在室温下超过1小时，每。 </li><li>应配备适当的过滤器，例如在BD LSR使用四个激光流式细胞仪获得的样品II。使用标准的流式细胞仪的校准和荧光补偿方法进行数据收集4。 </li></ol> 注意：不要设置5在收集过程中的散射或侧面散射阈值。 Trucount珠可以尽可能最低的低于阈值设置这些参数造成的一个子集珠在分析过程中不占。如果需要的仪器设置阈值，设置尽可能低的青色通道的阈值。因为CD45 +白细胞染色与面板将是青色，Trucount珠还发出荧光，在上午青色通道，这应该可以适当地收集所有相关数据。 <ol start="3"><li>涡样品管，持续5秒，然后再装上流式细胞仪。 Trucount珠荧光高度的多种渠道。在收集过程中发现高双阳性的人口是PE Cy5和APC（结肠门珠RS是最容易区分的珠子从细胞可能会有所不同，这取决于仪器）。选择您的停止门珠门，并记录数据，直到至少有20,000收购珠。 </li><li>使用合适的软件分析数据，作为FlowJo（Treestar，亚什兰，OR）等。 图1显示了用于分析不同的白细胞人口从有代表性的控制抽血的门控方案。 </li></ol> 5。 Trucount计算<ol><li>每个Trucount管含有荧光微球的冻干颗粒。在添加液体的管，和涡旋，珠粒应成为同样分布在整个样品。珠颗粒的数量略有不同批号和管的储物袋上可以找到。 </li><li>栅极Trucount珠和如在图1中所示的细胞群，以确定事件计数为每个人口。的事件的数量的比较我n的珠珠最初的总数在管的栅极连接到将允许您确定样品收集的，然后可以被用来确定的绝对浓度（ 即 ，细胞/微升），为每个人口的比率。可以使用下面的公式，用于此目的：细胞浓度（细胞/微升的全血）= [＃人口事件/（＃珠事件/＃总珠球团）] / 100微升。 </li></ol> 6。代表性的成果<img src="/files/ftp_upload/4302/4302fig1.jpg" alt="图1" fo:content-width="5.5in" fo:src="/files/ftp_upload/4302/4302fig1highres.jpg" /> 图1。使用的浇注方案的主要白细胞人口数据从健康志愿者代表进行分析。 A）Trucount珠（I）的门控，排除细胞。粒（二）所划定并的lympohcytes和单核细胞分为3 popluations：CD14阴性淋巴细胞（ⅲ），（ⅳ）所有CD14阴性细胞，和非淋巴细胞（ⅴ）。 B）CD14阴性细胞是门控区分CD4 +Ť细胞（六），CD8 + T细胞（七），乙细胞（八），CD56明亮的NK细胞（九），CD56昏暗的NK细胞（X），和CD56阴性NK细胞（十一）。 C）所有的CD14阴性细胞的门控来区分髓系（xii）及浆细胞（ⅹⅲ）的树突状细胞。 D）非，lympocytes的选通，区分非古典（十四），中级（十五），古典（十六）单核细胞。 <a href="http://www.jove.com/files/ftp_upload/4302/4302fig1large.jpg" target="_blank">点击此处查看大图</a> 。 使用我们提出的面 ​​板，也可以加以区分的更具体的，未示出在图1中（ 例如 ，NKT细胞或嗜中性粒细胞）的细胞亚群，和门控方案可以扩展或修改，以满足特定的学习需要。某些门控步骤所示，该方法是独特的。特别需要注意的，包括栅极和排除栅极周围绘制Trucount珠放置在另一个上面，一个栅极珠粒进行计数，和一个排除珠粒从蜂窝的分析（ 图1A）。此外，淋巴细胞，单核细胞和粒细胞，因为不容易被区分全血中，因为它们是在外周血单核细胞的前向散射和侧向散射光，浇注使用这些细胞CD45表达和侧向散射光往往是必要的（ 图1A）。在部分地块高CD16的表达（ 图1C和图1D）是有区别的，不能分离淋巴细胞和单核细胞CD45和侧向散射光的污染粒细胞（圆圈内）。污染的粒细胞的数量通常是很小的，并且它们不干扰与单核细胞和NK细胞的门控。

<ol>
	<li>Taylor, M. J., London, N. J., Thirdborough, S. M., Lake, S. P., James, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cryobiology+of+rat+and+human+dendritic+cells:+preservation+and+destruction+of+membrane+integrity+by+freezing.">The cryobiology of rat and human dendritic cells: preservation and destruction of membrane integrity by freezing.</a> Cryobiology. 27, 269-278 (1990).</li><li>Reimann, K. A., Chernoff, M., Wilkening, C. L., Nickerson, C. E., Landay, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+lymphocyte+immunophenotype+and+proliferative+responses+in+cryopreserved+peripheral+blood+mononuclear+cells+from+human+immunodeficiency+virus+type+1-infected+donors:+implications+for+multicenter+clinical+trials.+The+ACTG+Immunology+Advanced+Technology+Laboratories.">Preservation of lymphocyte immunophenotype and proliferative responses in cryopreserved peripheral blood mononuclear cells from human immunodeficiency virus type 1-infected donors: implications for multicenter clinical trials. The ACTG Immunology Advanced Technology Laboratories.</a> Clin Diagn Lab Immunol. 7, 352-359 (2000).</li><li>Boonlayangoor, P., Telischi, M., Boonlayangoor, S., Sinclair, T. F., Millhouse, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+human+granulocytes:+study+of+granulocyte+function+and+ultrastructure.">Cryopreservation of human granulocytes: study of granulocyte function and ultrastructure.</a> Blood. 56, 237-245 (1980).</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, 1522-1530 (2006).</li><li>Brando, B., Barnett, D., Janossy, G., Mandy, F., Autran, B., Rothe, G., Scarpati, B., D'Avanzo, G., D'Hautcourt, J. L., Lenkei, R., Schmitz, G., Kunkl, A., Chianese, R., Papa, S., Gratama, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytofluorometric+methods+for+assessing+absolute+numbers+of+cell+subsets+in+blood.+European+Working+Group+on+Clinical+Cell+Analysis.">Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis.</a> Cytometry. 42, 327-346 (2000).</li><li>Bull, M., Lee, D., Stucky, J., Chiu, Y. L., Rubin, A., Horton, H., McElrath, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+blood+processing+parameters+for+optimal+detection+of+cryopreserved+antigen-specific+responses+for+HIV+vaccine+trials.">Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials.</a> J. Immunol Methods. 322, 57-69 (2007).</li><li>Kantor, A. B., Roederer, M., Herzenberg, L., Blackwell, C., Weir, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The handbook of Experimental Immunology. 43, 1-49 (1996).</li><li>Hulspas, R. <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+and+the+stability+of+phycoerythrin-tandem+dye+conjugates.">Flow cytometry and the stability of phycoerythrin-tandem dye conjugates.</a> Cytometry A. 75, 966-972 (2009).</li><li>Le Roy, C., Varin-Blank, N., Ajchenbaum-Cymbalista, F., Letestu, R. <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+APC-tandem+dyes+are+degraded+through+a+cell-dependent+mechanism.">Flow cytometry APC-tandem dyes are degraded through a cell-dependent mechanism.</a> Cytometry A. 75, 882-890 (2009).</li><li>Mandy, F., Brando, B. <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+absolute+cell+counts+using+immunophenotypic+techniques.">Enumeration of absolute cell counts using immunophenotypic techniques.</a> Curr. Protoc. Cytom. Chapter 6, Unit 6 (2001).</li><li>Vuckovic, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+dendritic+cells+in+clinical+practice+using+a+new+whole+blood+single-platform+TruCOUNT+assay.">Monitoring dendritic cells in clinical practice using a new whole blood single-platform TruCOUNT assay.</a> J. Immunol Methods. 284, 73-87 (2004).</li><li>Lichtner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+dendritic+cells+and+interferon-alpha+production+in+patients+with+tuberculosis:+correlation+with+clinical+outcome+and+treatment+response.">Circulating dendritic cells and interferon-alpha production in patients with tuberculosis: correlation with clinical outcome and treatment response.</a> Clin. Exp. Immunol. 143, 329-337 (2006).</li><li>Hosmalin, A., Lichtner, M., Louis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+analysis+of+dendritic+cell+subsets:+the+dendritogram.">Clinical analysis of dendritic cell subsets: the dendritogram.</a> Methods Mol. Biol. 415, 273-290 (2008).</li></ol>

没有利益冲突的声明。

在这份报告中，我们提出了一个珠列举流式细胞仪新鲜全血中的白细胞人口为基础的方法，并覆盖所需的参数的多中心临床试验中使用的集中样品分析。这种方法的基础上，优化的BD Trucount协议，并允许其可靠的多中心临床试验中使用的设置。染色法很简单，大约需要45分钟来执行，使血液处理实验室技术人员来执行它，并冻结和运送样本到中央分析实验室进行分析是可行的。该试验仅需要100微升...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> Trucount绝对计数管</td><td> BD Biosciences公司</td><td> 340334 </td></tr><tr><td> 10X FACS裂解解决方案</td><td> BD Biosciences公司</td><td> 349202 </td></tr><tr><td> B类及豁免的运输系统，绝缘</td><td> SAF-T-PAK </td><td> STP-320 </td></tr><tr><td>单克隆抗体CD45 AmCyan </td><td> BD Biosciences公司</td><td> 339192 </td></tr><tr><td> CD3 FITC单克隆抗体</td><td> BD Biosciences公司</td><td> 349201 </td></tr><tr><td> CD8 PerCp-CY 5.5单克隆抗体</td><td> BD Biosciences公司</td><td> 341051 </td></tr><tr><td> CD4的Alexa Fluor 700单克隆antibo的DY </td><td> BD Biosciences公司</td><td> 557922 </td></tr><tr><td> HLA-DR ECD单克隆抗体</td><td> Beckman Coulter公司</td><td> IM3636 </td></tr><tr><td>单克隆抗体CD14 V450 </td><td> BD Biosciences公司</td><td> 560349 </td></tr><tr><td>单克隆抗体CD19 PE </td><td> BD Biosciences公司</td><td> 555413 </td></tr><tr><td> CD16 APC-H7单克隆抗体</td><td> BD Biosciences公司</td><td> 560195 </td></tr><tr><td> CD56 PE-CY7单克隆抗体</td><td> BD Biosciences公司</td><td> 335791 </td></tr><tr><td>表面CD11c APC单克隆抗体</td><td> BD Biosciences公司</td><td> 559877 </td></tr><tr><td> CD123 PE-Cy5标记的单克隆抗体</td><td> BD Biosciences公司</td><td> 551065 </td></tr></tbody></table>

enumeration of major peripheral blood leukocyte populations for multicenter clinical trials using a whole blood phenotyping assay

外周血白细胞的超低温保存的被广泛用来保存在临床试验中的细胞的免疫应答的评估，为便于和标准化的免疫学评估提供了许多优点，但在此过程中的有害​​影响已被观察到一些细胞亚群，如粒细胞，B细胞和树突状细胞1-3。含量测定新鲜的白 ​​细胞在体内的细胞状态，给出了更准确的图片，但往往是难以执行的背景下，大规模的临床试验。新鲜的细胞检测是依赖于志愿者的承诺和时限，如果费时，他们的应用程序可以是不切实际的，由于实验室工作人员的工作时间要求。此外，在多个中心进行试验时，与要执行的检测的资源和培训实验室可能不会被位于足够接近临床站点。为了解决这些问题，我们已经开发开发了一个11-色抗体染色面板，可以使用Trucount管（Becton Dickinson公司，圣何塞，加利福尼亚州）的表型和外周血内列举主要的白细胞种群，得到更强大的细胞类型特异性的信息比检测诸如全血细胞计数（CBC）或分析与市售面板设计Trucount管，染色只有少数细胞类型。染色过程很简单，只需要100微升新鲜全血，需时约45分钟，使其成为可行的标准​​的血液处理实验室执行。它改编自BD Trucount管技术数据表（ <a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">8/2010</a> ）。在中央检测实验室，可以在散装提前准​​备染色的抗体鸡尾酒，运到现场处理实验室。可以是固定的彩绘管冻结装运中央多色流式细胞仪分析检测实验室。从这种染色面板生成的数据可以被用来追踪干预有关的白细胞浓度随着时间的推移的变化，并可以很容易地得到进一步的发展，以评估特定细胞类型的兴趣的激活状态。在这份报告中，我们展示了使用血液处理实验室技术人员进行染色新鲜的全血和步骤来分析这些染色样品在中央检测实验室支持的多中心临床试验的程序。视频详细信息的过程，因为它是在艾滋病疫苗试验网络（HVTN）的临床试验抽血的背景下进行。

Watch this Scientific Journal Video about Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay at JoVE.com

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

在这份报告中，我们展示了染色和分析步骤，新鲜全血进行列举主要的先天免疫和适应性白细胞种群的表型分析。执行这些程序在多中心临床试验的背景下，我们强调的注意事项。

枚举主要外周血白细胞人口多中心临床试验，使用全血表型分析

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

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JoVE Journal

Immunology and Infection

14.9K 次观看.  Fred Hutchinson Cancer Research Center. 在这份报告中，我们展示了染色和分析步骤，新鲜全血进行列举主要的先天免疫和适应性白细胞种群的表型分析。执行这些程序在多中心临床试验的背景下，我们强调的注意事项。

视频: Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

补消耗的特定细胞群的枯竭

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

科研

免疫与感染

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

特定细胞群的净化荧光激活细胞分选（FACS）

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

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 ...

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  &amp;#947;&amp;#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 ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

一个功能全血含量测量分枝杆菌的生存能力，记者基因标记卡介苗或M. TB（卡介苗勒克斯 / M. TB勒克斯）

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

在富含白细胞的血液样本卤化过氧化物酶活性的快速，具体的评估

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

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.

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

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 ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

大鼠外周全血细胞免疫表型和因子生成的单细胞分析

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...