Name: isolation and flow cytometric analysis of human endocervical gamma delta t cells
Uploaded: 2017-02-06T10:30:00.000+00:00
Duration: 8 min 10 s
Description: Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells at JoVE.com

我们感谢迈阿密WIHS研究的参与者为自己愿意参加这项工作。在这个手稿的数据是由迈阿密妇女间艾滋病毒研究（WIHS）收集。本出版物的内容完全是作者的责任，并不代表美国国立卫生研究院（NIH）的官方意见。这项工作是由过敏和传染病研究所和妇女间的艾滋病毒感染的研究（WIHS）的授权号U01 AI103397]，国家过敏和传染病研究所，卫生部[基金号P30AI073961]的国家机构，国家中心推进支持生转化科学与健康[授权编号UL1TR000460和1KL2TR000461]研究所国立少数民族健康与健康差距，与儿童健康和人类发展研究所研究院[授权号码K23HD074489。

研究活动发生在迈阿密的HIV研究单位的大学与迈阿密妇女HIV间研究（WIHS），并事先获得批准，迈阿密艾滋病研究中心（CFAR）.Institutional审查委员会（医学迈阿密大学米勒学院）合作招聘和任何评估或研究相关手续。 1.宫颈刷样品采集注:参与者接受由经过培训的医师或妇科医生和上皮内淋巴细胞通过插入细胞刷下窥器检查进行收集进行阴道检查。 <ol><li> 参与者特性 <ol><li>招谁是年龄在18岁至45岁的性活跃，不怀孕，不避孕药物或使用宫内节育器的妇女参加。 </li><li>阴道检查之前，必须接受参与者在绿色10毫升抽血顶肝素真空采血管为外周血单核细胞（PBMC）中分离。以作为宫颈上皮内分析9的控制。 </li></ol></li><li> 宫颈样本采集 <ol><li>插入适当大小的无菌窥器成无润滑阴道。使用温水，以促进窥器和无菌纱布擦拭黏液的插入。窥器的位置允许操作系统和宫颈的完全可视化。 </li><li>插入宫颈刷入宫颈管，直到只有最靠近手刷毛是可见的。 360°刷按顺时针方向旋转。转移刷入含有5ml IMDM培养基的15毫升管中。 </li><li>收集后，立即将装有冰块细胞刷管。为了获得收集上皮细胞的活力较高，1小时内交付细胞刷到实验室和过程。 </li></ol></li></ol><ol><li>采用含4个短（10秒）招宫颈细胞刷涡流管。 </li><li>离心管，含有细胞刷，在250×g的10分钟。 </li><li>小心地取出从管的细胞刷（请勿打扰在管底部的沉淀），并添加IMDM媒体1毫升，并将管在冰上。 </li><li>放置在50ml管中的顶部100mm的细胞过滤的细胞刷。 </li><li>洗涤用20毫升的IMDM细胞刷。 </li><li>离心管在250×g下10分钟。 </li><li>倒出上清液和颗粒细胞悬浮于1mL的IMDM并与在2.3中描述的已重悬浮沉淀结合。 </li><li>计数使用自动细胞计数器10中的细胞或通过细胞悬浮液的台盼蓝染料的10微升合并10μL的计数使用血球细胞。轻轻吸管上下10次以混合细胞和染料。装载10μL的混合物制成的任两个血细胞计数腔室的开口。算在五格区的所有细胞。保持存活和非存活细胞的单独计数。使用下面的公式确定细胞存活率:％存活细胞=非存活细胞×100 /总细胞数的细胞。 确定使用下面的计算每毫升随后的细胞浓度（和细胞的总数）。每毫升总细胞=总细胞计数X（稀释因子/计数平方数）×10000个细胞/ ml的。 </li><li>冻结使用含有10％的DMSO和90％FBS的冷冻介质分离的子宫颈细胞。 </li></ol> 3.流式细胞仪<ol><li>重悬在100μL的FACS缓冲液1-3×10 5个颈管细胞（含有1％牛血清白蛋白的PBS </li><li>加入1微升/ 10 6个活/死可固定黄色死细胞染色试剂盒以及用于以表1记载的浓度的表面标志的抗体。 </li><li>孵育细胞在+ 4℃，30分钟，避光。 </li><li>添加1毫升FACS缓冲液和细胞，并离心管5分钟，在350 xg离心</li><li>重悬在300微升1％多聚甲醛的细胞沉淀。 </li><li>准备使用通过加入FACS缓冲液100微升和珠粒一滴和用于样品染色的抗体的相同浓度用于染色珠和抗体补偿控制。 </li><li>获取有关流式细胞仪，配备405纳米，488纳米，和635nm激光流染色珠和样品。 </li></ol> 4.宫颈的γδT细胞分离<ol><li> 磁珠分离 <ol><li>重悬宫颈细胞沉淀（≤107总细胞），在2.6所述，在包括在材料与试剂的表中引用的T细胞受体的γ增量微珠试剂盒缓冲液40微升。 </li><li>遵循两步染色方案套件说明:第一，才子H +反-TCR伽玛三角洲半抗原抗体和第二，具有抗半抗原微-FITC </li><li>在4-8℃下温育15分钟后，在300 xg离心加入1毫升缓冲液和离心10分钟清洗菌体。颠倒离心管弃去上清液，挖掘干燥，一张纸的帮助。 </li><li>重悬在提供缓冲的500微升细胞沉淀并通过根据供应商的协议将其放置在适当的磁分离器制备的磁列。 </li><li>应用细胞悬液上柱，收集穿过未标记细胞并用缓冲液适量（500微升）洗柱3倍。通过添加缓冲三次，每次一旦列储为空的时间执行清洗步骤。共收集污水。这是未标记的，阴性细胞级分</li><li>删除该列形成磁分离器和吸管1毫升缓冲液上柱并立即与磁性标签冲出分数用力将与列提供的柱塞编细胞。 注:孤立的γδT细胞已经荧光染色流式细胞分析，因为磁性标记的抗半抗原微珠用荧光染料FITC结合。 </li></ol></li><li> 细胞分选 <ol><li>对于细胞分选，标签颈管细胞活力模和抗体面板在第3节中所述。 </li><li>设置电子门的实况，CD45 + CD3 +和γ三角洲阳性细胞并进行细胞分选。 </li></ol></li></ol>

<ol>
	<li>Kaul, R., 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+genital+tract+immune+milieu:+an+important+determinant+of+HIV+susceptibility+and+secondary+transmission.">The genital tract immune milieu: an important determinant of HIV susceptibility and secondary transmission.</a> J Reprod Immunol. 77, 32-40 (2008).</li><li>Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+early+infection+to+prevent+HIV-1+mucosal+transmission.">Targeting early infection to prevent HIV-1 mucosal transmission.</a> Nature. 464, 217-223 (2010).</li><li>Hayday, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma][delta]+cells:+a+right+time+and+a+right+place+for+a+conserved+third+way+of+protection.">gamma][delta] cells: a right time and a right place for a conserved third way of protection.</a> Ann Rev Immunol. 18, 975-1026 (2000).</li><li>Nakasone, C., 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=Accumulation+of+gamma/delta+T+cells+in+the+lungs+and+their+roles+in+neutrophil-mediated+host+defense+against+pneumococcal+infection.">Accumulation of gamma/delta T cells in the lungs and their roles in neutrophil-mediated host defense against pneumococcal infection.</a> Microbes and infection / Institut Pasteur. 9, 251-258 (2007).</li><li>Autran, 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=T+cell+receptor+gamma/delta++lymphocyte+subsets+during+HIV+infection.">T cell receptor gamma/delta+ lymphocyte subsets during HIV infection.</a> Clin Exp Immunol. 75, 206-210 (1989).</li><li>Pauza, C. D., Poonia, B., Li, H., Cairo, C., Chaudhry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gammadelta+T+Cells+in+HIV+Disease:+Past,+Present,+and+Future.">gammadelta T Cells in HIV Disease: Past, Present, and Future.</a> Front Immunol. 5, 687 (2014).</li><li>Hayday, A. C., Spencer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barrier+immunity.">Barrier immunity.</a> Sem Immunol. 21, 99-100 (2009).</li><li>Cheroutre, H., Lambolez, F., Mucida, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+light+and+dark+sides+of+intestinal+intraepithelial+lymphocytes.">The light and dark sides of intestinal intraepithelial lymphocytes.</a> Nature Rev Immunol. 11, 445-456 (2011).</li><li>Strbo, N., 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=Loss+of+Intra-Epithelial+Endocervical+Gamma+Delta+(GD)+1+T+Cells+in+HIV-Infected+Women.">Loss of Intra-Epithelial Endocervical Gamma Delta (GD) 1 T Cells in HIV-Infected Women.</a> AJRI. 75, 134-145 (2016).</li><li>Szabo, S. E., Monroe, S. L., Fiorino, S., Bitzan, J., Loper, K. <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+an+automated+instrument+for+viability+and+concentration+measurements+of+cryopreserved+hematopoietic+cells.">Evaluation of an automated instrument for viability and concentration measurements of cryopreserved hematopoietic cells.</a> Lab Hematol. 10, 109-111 (2004).</li><li>Alcaide, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Vaginosis+Is+Associated+with+Loss+of+Gamma+Delta+T+Cells+in+the+Female+Reproductive+Tract+in+Women+in+the+Miami+Women+Interagency+HIV+Study+(WIHS):+A+Cross+Sectional+Study.">Bacterial Vaginosis Is Associated with Loss of Gamma Delta T Cells in the Female Reproductive Tract in Women in the Miami Women Interagency HIV Study (WIHS): A Cross Sectional Study.</a> PloS one. 11, e0153045 (2016).</li><li>Masson, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+genital+tract+cytokine+signatures+of+sexually+transmitted+infections+and+bacterial+vaginosis+in+women+at+high+risk+of+HIV+infection:+a+cross-sectional+study.">Defining genital tract cytokine signatures of sexually transmitted infections and bacterial vaginosis in women at high risk of HIV infection: a cross-sectional study.</a> Sex Transm Dis. 90, 580-587 (2014).</li><li>Fichorova, R. N., 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=Interleukin+(IL)-1,+IL-6,+and+IL-8+predict+mucosal+toxicity+of+vaginal+microbicidal+contraceptives.">Interleukin (IL)-1, IL-6, and IL-8 predict mucosal toxicity of vaginal microbicidal contraceptives.</a> Biol Reprod. 71, 761-769 (2004).</li><li>Juno, J. A., Boily-Larouche, G., Lajoie, J., Fowke, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection,+isolation,+and+flow+cytometric+analysis+of+human+endocervical+samples.">Collection, isolation, and flow cytometric analysis of human endocervical samples.</a> JoVE. , (2014).</li><li>McKinnon, L. R., 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=Optimizing+viable+leukocyte+sampling+from+the+female+genital+tract+for+clinical+trials:+an+international+multi-site+study.">Optimizing viable leukocyte sampling from the female genital tract for clinical trials: an international multi-site study.</a> PloS one. 9, e85675 (2014).</li><li>Dominguez, M. 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=Highly+multiplexed+quantitation+of+gene+expression+on+single+cells.">Highly multiplexed quantitation of gene expression on single cells.</a> J Immunol Methods. 391, 133-145 (2013).</li></ol>

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女性下生殖道粘膜脆弱性评价是临床研究解决艾滋病毒的采集和传播风险的重要组成部分。通常情况下FGT感染艾滋病毒是由生殖器分泌物（细胞因子，趋化因子和抗菌肽）12，13测量可溶性免疫调节剂进行评估。然而，这些生物标志物只是象征性阴道炎症，由生理和病理的事件受到影响，而且并不总是代表直接粘膜损伤。为了提高我们的粘膜因素如何增加获取和传播艾滋病病毒的风险的认识，黏膜损伤的细胞标记需要开发。 GD上皮内的T细胞存在于FGT，插上皮细胞之间，并且被认为有助于动态平衡以及对环境挑战的防御的第一道防线。 GD T细胞有POTEN<html> TiAl基合金构成额外的生物标志物，以评估FGT粘膜漏洞。 在这个协议中，示出了如何从人宫颈细胞刷样本制备上皮内淋巴细胞和易于掌握。这个过程的最关键的一点是通过样品的快速处理，以及通过在整个过程中保持细胞在冰上以保持细胞生存力。细胞产量将取决于细胞刷采集技术，处理技巧以及感染状态（HIV感染或其他病毒感染，细菌或真菌感染）。我们的团队和其他人发表了描述人类生殖道9，14，15种不同的淋巴细胞群的各种研究。在我们的手中，人们宫颈细胞刷样本可以提供0.5-5×10 6细胞用80-90％的生存能力。与常用的隔离方法相比类="外部参照"&gt; 14，15，由我们的方法的可行性和细胞产量增加。 这种方法提供了一个强大的和高效率的工具来研究子宫颈上皮内的γδT淋巴细胞和可以提供深入了解感染和/或炎症过程中迁移到这个车厢其他免疫细胞。粘膜组织的体内细胞组合物通常很难以全面和定量的方式进行调查。技术如免疫组织化学和流式细胞术是由抗原特异性的单克隆抗体的可用性和由小数目并行测量的，可以在每个单独的细胞来进行限定。传统的高通量测定法，如基因表达阵列，在整个组织中进行时，提供平均基因表达水平的信息，并且可以仅间接相关的蜂窝亚群数量的修改。这些限制变得特别困难的学习少数民族人口，或者说缺乏专属标记的细胞群时需要克服。通过组合"荧光激活细胞分选"（FACS）和"单细胞PCR基因表达分析"一可以执行上皮内GD T细胞在宫颈的高通量转录分析。这种方法与使用微流体技术的利用现代流式细胞仪的各个单个细胞用准确度和精密度排序的能力，一起从mRNA的微量预型高灵敏度多元PCR，由此允许高达96个基因的表达的平行分析对于每个单独的小区16。

我们开发使用宫颈刷样本，以评估上皮内GD T细胞的方法。宫颈管被柱状上皮单层衬里。上皮内淋巴细胞代表驻留在上皮细胞层内前线淋巴细胞，可以迅速地在遇到病原微生物7,8引发免疫反应。了解宫颈上皮内舱的免疫活动是有效的战略设计，以防止伤口感染，包括艾滋病毒非常重要的。 我们的方法可应用于新鲜收集的人类宫颈样品以及冷冻颈管细胞进一步探索上皮内GD T细胞的妇女感染艾滋病毒或感染艾滋病风险的作用。该协议的主要目标是发现新的免疫活动在宫颈上皮内舱，以及evaluatËGD T细胞应答粘膜脆弱的妇女感染艾滋病毒的标志。 在周围FGT免疫知识的大量间隙部分是由于难以在成功地收集和处理的粘膜样品。此外，粘膜免疫细胞的复杂的异质性，以及它们与彼此相互联系，都是以确定反映免疫系统的状态和能力临床相关的测量的主要挑战。我们开发了一种方法，以获得高度纯化的上皮内GD的T细胞，可以进一步使用可用于识别的FRT免疫力的基本机制临界高度多路复用，单细胞的技术进行分析。

<table><tbody><tr><td>Cytobrush Plus GT&amp;nbsp;</td><td>(CareFusion, San Diego, CA, USA</td><td></td><td></td></tr><tr><td>IMDM,Iscove&#039;s Modified Dulbecco&#039;s Medium&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>12440061</td><td></td></tr><tr><td>Beckman Coulter automated cell counter (The Vi-CELL Series Cell Viability Analyzer</td><td>Beckman Coulter</td><td>496178</td><td></td></tr><tr><td>DMSO, Dimethyl sulfoxide&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>D2650</td><td></td></tr><tr><td>FBS, Fetal Bovine Serum</td><td>Invitrogen, Gibco</td><td>26140-079</td><td></td></tr><tr><td>PBS</td><td>Invitrogen, Gibco</td><td>10010023</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A-9647</td><td></td></tr><tr><td>LIVE/DEAD Fixable Yellow Dead Cell Stain kit&amp;nbsp;</td><td>Life Technologies,</td><td>L349S9</td><td></td></tr><tr><td>1% paraformaldehyde</td><td>Sigma Aldrich&amp;nbsp;</td><td>D6148</td><td></td></tr><tr><td>Fortessa flow cytometer&amp;nbsp;</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>UltraComp eBeads</td><td>eBioscience</td><td>01-2222-42</td><td></td></tr><tr><td>ArC Amine Reactive Compensation Beads (Life Technologies, Grand Island, NY, USA)</td><td>Life Technologies</td><td>A10346</td><td></td></tr><tr><td>anti-TCR gamma/delta MicroBead Kit&amp;nbsp;</td><td>Milteny Miltenyi Biotec Inc.</td><td>130-050-701</td><td></td></tr><tr><td>MS column</td><td></td><td>103-042-201</td><td></td></tr><tr><td>MACS separator</td><td></td><td>4124</td><td></td></tr></tbody></table>

isolation and flow cytometric analysis of human endocervical gamma delta t cells

女性生殖道（FRT）粘膜免疫系统作为防御的第一道防线。生殖器粘膜更好地了解因此对于理解不同的病原体，包括病毒的致病性是必不可少的。伽马增量（GD）T细胞是"非常规的"T细胞的原型，并代表其γ和δ链组成的异二聚体T细胞受体（TCR）的表达定义的T细胞的相对小的子集。此它们区别于由α-β的TCR限定的经典和更好的已知CD4 +辅助T细胞和CD8 +细胞毒性T细胞。 GD T细胞常表现出组织特异性定位及上皮充实。 GD T细胞协调在炎症，肿瘤监视，感染性疾病，和自身免疫的免疫应答。 在这里，我们提出可重复分离和分析人的子宫颈上皮内GD T淋巴细胞的方法。我们公顷从已经参加的女性妇女间HIV感染研究（WIHS）中使用的宫颈细胞刷样本。在条件有关GD T细胞相互作用的知识，其中有侮辱了阴道粘膜可应用于任何临床研究中，粘膜漏洞被解决，包括阴道microbicides.In另外的发展，对粘膜GD T细胞应答的知识有潜力在治疗感染性疾病的应用GD牛逼细胞为基础的免疫疗法。

最近，我们分析了子宫颈上皮内GD T细胞，我们是第一个报告关于宫颈的γδT细胞的艾滋病病毒感染的妇女9,11损失。这里，我们描述了协议来分离和分析宫颈的γδT细胞的子集。正如图1所示，GD的T细胞可以容易地在人宫颈细胞样品中检测到。 使用multiparametar流式细胞术为基础的免疫（ 图1）进行了说明宫颈伽马增量V1（GD1）T细胞的独特群体。艾滋病毒感染宫颈样本的代表性门控策略，如图1所示。通过设置正向/侧向散射（ 图1A）的电子门随后用t上的总颈管细胞进行分析他排除细胞的双峰（ 图1B）。活细胞用活/死可固定黄胺死细胞染色（ 图1C）的死细胞的排斥所定义。于活门控细胞（ 图1D）中，随后CD3表达的分析（ 图1E）进行CD45的表达的分析。伽马增量1（GD1）或伽玛增量2（GD2）的阳性细胞被定义为表达GD TCR（TCR伽玛三角洲V1或T细胞受体的γ增量V2），CD3 +的细胞亚群。此外，证实只有CD3 + T细胞表达，因为门控CD3 T细胞受体伽马三角洲- T细胞不表达TCR伽玛三角洲V1或delta V2。门控CD3 + GD1 +揭示细胞（ 图1G）的表型分析，即大多数GD1细胞（〜74％）的CD4 - CD8 - 。 在本研究中，利用endoce的可行性rvical刷样品纯化的上皮内GD的T细胞通过正磁选择被证明（ 图2）。 <img alt="图1" src="/files/ftp_upload/55038/55038fig1.jpg" /> 图 1: 通过流式细胞仪分析宫颈粘膜伽玛三角洲（GD）T细胞亚群。以下门控策略被用于确定宫颈T细胞群:根据大小和粒度（FSC，前侧散射VS×SSC，侧散射）（A）的子宫颈细胞刷细胞进行门控。 （B）细胞双峰被设置VS在FSC-A的电子门（区）排除SSC-W（宽）。 （C）活细胞被门上的可行性染料阴性人群。 （ 四 ）电子门是活，CD45阳性细胞集。 （ 五 ）电子门的CD45阳性细胞被设置CD3阳性细胞（F）TCRVD1（GD1）和TCRVD2（GD2）表达分析了CD3 +细胞。 （G），CD4和CD8的表达上分析GD1 +细胞。 （H）TCRVD1和TCRVD2表达上分析门控CD3阴性细胞。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55038/55038fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55038/55038fig2.jpg" /> 图2: 宫颈管的γδT细胞（GD）通过积极磁选的分离。宫颈GD T细胞的频率前后正磁分离得到了证实。子宫颈细胞首先用抗TCR伽玛三角洲抗体，然后用荧光与抗半抗原微珠-FITC染色。 TCR GD + T细胞的百分比之前以及m后报agnetic分离。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55038/55038fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells at JoVE.com

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

人类宫颈的γδT细胞的分离和流式细胞仪分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

isolation-flow-cytometric-analysis-human-endocervical-gamma-delta-t

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

8.7K 次观看.  Miller School of Medicine, University of Miami. We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

视频: Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

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 ...

科研

免疫与感染

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

收集，分离，和人类宫颈样品的流式细胞仪分析

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

医学

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

分离和<em&gt;离体</emVδ1的&gt;企业文化<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt;γδT细胞，胸腺外的αβT细胞祖

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

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 ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

通过成像流式细胞仪人单核细胞来源的树突状细胞的特征:两个单核细胞分离方法的比较

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and activity of cytotoxic T cells within tumors and their tumor microenvironment (TME) are now well-established prognostic and predictive biomarkers for numerous tumor types. However, it is well-known that various tumor treatment modalities, including radiotherapy, chemotherapy, immunotherapy, and targeted therapy, modulate not only the immunogenicity of the tumor but also the immune system itself. Consequently, the interaction between tumor cells and T cells requires more intensive study in different therapeutic contexts to fully understand the complex role of T cells during tumor therapy. To address this need, a protocol was developed to analyze the activity and proliferative capacity of human cytotoxic (CD8+) T cells in co-culture with pre-treated tumor cells. Specifically, CD8+ T cells from healthy donors are stained with the non-toxic proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated using CD3/CD28-coated plates. Subsequently, T cells are co-cultured with pre-treated tumor cells. As a readout, T cell proliferation is quantified by measuring CFSE signal distribution and assessing the expression of surface activation markers via flow cytometry. This can be further complemented by quantifying cytokine release using enzyme-linked immunosorbent assay (ELISA). This method facilitates the evaluation of treatment-induced changes in the interaction between tumor cells and T cells, providing a foundation for more detailed analyses of tumor treatment modalities and their immunogenicity in a human ex vivo setting. Additionally, it contributes to the reduction of pre-clinical in vivo analyses.

The present protocol describes an experimental workflow that allows for the ex vivo analysis of human T cell stimulation in an allogenic co-culture system with pre-treated tumor cells.

在预处理肿瘤细胞的同种异体共培养环境中分析人 T 细胞活性

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and ...

癌症研究

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

人雌性生殖道树突状细胞表型鉴定的分离及功能研究

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

双氟铬流式细胞仪对七种免疫细胞子集的判别

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

分离子宫先天淋巴细胞，用于流式细胞术分析

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the lymph node microenvironment in B-cell non-Hodgkin&#39;s lymphoma is analyzed. Subsets of circulating T-follicular helper cells (cTfh), the circulating memory compartment of Tfh cells in the blood, are also perturbed in disease and therefore represent potential novel predictive biomarkers. Peripheral blood-based testing is advantageous because it is relatively non-invasive and allows simple serial monitoring.This article describes a method for isolating CD4+ T-cells from human blood, and further analysis by flow-cytometry to enumerate cTfh cells and the proportions of their various subsets (cTfhPD-1-/+/hi, cTfh1,2,17 and cTfh1/17). The level of these subsets was then compared between normal subjects and patients with lymphoma. We found that the method was robust enough to obtain reliable results from routinely collected patient material. The technique we describe for the analysis can be easily adapted to cell sorting and downstream applications such as RT-PCR.

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Here, a protocol for the isolation and characterization of CD4+ T-cell subsets from human peripheral blood is described. Purified CD4+ T-cells are analyzed by flow cytometry to determine proportions of T-follicular helper cell subsets.

cd4+ t 细胞的分离及6色流式细胞仪分析外周血中循环 t-滤泡 (ctfh) 细胞亚群

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the ...