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>

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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 border="0" cellpadding="0" cellspacing="0" width="648">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cytobrush Plus GT&#160;</td>
			<td style="width:147px;">(CareFusion, San Diego, CA, USA</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">IMDM,Iscove&#39;s Modified Dulbecco&#39;s Medium&#160;</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td align="right" style="width:119px;">12440061</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Beckman Coulter automated cell counter (The Vi-CELL Series Cell Viability Analyzer</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">496178</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO, Dimethyl sulfoxide&#160;</td>
			<td style="width:147px;">Sigma Aldrich&#160;</td>
			<td style="width:119px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS, Fetal Bovine Serum</td>
			<td style="width:147px;">Invitrogen, Gibco</td>
			<td style="width:119px;">26140-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:147px;">Invitrogen, Gibco</td>
			<td align="right" style="width:119px;">10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BSA</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">A-9647</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LIVE/DEAD Fixable Yellow Dead Cell Stain kit&#160;</td>
			<td style="width:147px;">Life Technologies,</td>
			<td style="width:119px;">L349S9</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1% paraformaldehyde (Sigma-Aldrich</td>
			<td style="width:147px;">Sigma Aldrich&#160;</td>
			<td style="width:119px;">D6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fortessa flow cytometer&#160;</td>
			<td style="width:147px;">Becton Dickinson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UltraComp eBeads</td>
			<td style="width:147px;">eBioscience</td>
			<td style="width:119px;">01-2222-42</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">ArC Amine Reactive Compensation Beads (Life Technologies, Grand Island, NY, USA)</td>
			<td style="width:147px;">Life Technologies</td>
			<td style="width:119px;">A10346</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-TCR gamma/delta MicroBead Kit&#160;</td>
			<td style="width:147px;">Milteny Miltenyi Biotec Inc.</td>
			<td style="width:119px;">130-050-701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MS column</td>
			<td style="width:147px;"></td>
			<td style="width:119px;">103-042-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACS separator</td>
			<td style="width:147px;"></td>
			<td align="right" style="width:119px;">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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

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

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

迁移人T淋巴细胞分离，培养和分析<em在体外</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

科研

免疫与感染

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

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 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

小鼠小肠淋巴细胞的分离和流式细胞表征

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

粒子结合Bet v 1的变应原PM10的流式细胞分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

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 ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...