这个项目是由美国国立卫生赠款HL087088（MK）和HL18208（MK）的支持。

1。人T淋巴细胞的分离<ol><li>人体血液中获得一个健康的捐赠者。让血液冷却至室温（约30分钟），然后再继续下一步。 </li><li>到8 ml圆底聚苯乙烯管轻轻吸液管3毫升室温变形密度梯度介质。轻轻地加入3毫升全血的变形媒体上。重要的是要避免两种试剂混合。 </li><li> 500在室温下45分钟XG的离心管。 </li><li>外周血单个核细胞（PBMC）的离心后，现在已经从其他血液成分分离到顶部的细胞层。外周血单个核细胞层的出现，从上往下，作为第一个阴天乐队。 </li><li>小心取出的血液明显的黄色上相以上的外周血单个核细胞层，然后使用P1000的微量外周血单个核细胞层转移到15 mL或50 mL锥形管。 </li><li>用PBS洗两次PBMC的细胞，每次5分钟，离心500 XG。每次冲洗后，上清液将会有所阴天。 </li></ol> 2。文化对人T淋巴细胞<ol><li>使用吸管，在20毫升含10％胎牛血清的RPMI 1640媒体传输到T - 75培养瓶中的外周血单个核细胞，青霉素/链霉素，1％和1μg/ mL的植物血凝素（PHA）。 </li><li>在37 ° C和5％CO 2至少1小时，最长为24小时。这一步使单核细胞，将附着在烧瓶表面，仍然处于悬浮状态的淋巴细胞分离。如果在这一步一个潜伏期短（1小时），它是可以接受的RPMI 1640培养液含10％胎牛血清和1％的青霉素/链霉素2.1步中指定的补充没有与PHA的媒体。 </li><li>小心取出所有的媒体都从瓶中，加入50 mL锥形管，并在500 XG离心5分钟。 </li><li>悬浮细胞沉淀，目前主要包含淋巴细胞，并转移到一个新的T - 75烧瓶中含有25毫升的RPMI 1640介质含10％胎牛血清的细胞，1％青霉素/链霉素，1微克/毫升PHA的。 </li><li>在37 ° C孵育3天，如果最初的PBMC培养过夜（2天）。经过24小时的增长，它可能需要添加15-20毫升新鲜媒体，并转移到一个较大的T - 175烧瓶。 </li><li>经过3天，用吸管取出烧瓶并转移至50 mL锥形管媒体和暂停淋巴细胞。在500 XG离心5分钟。 </li><li>重悬细胞沉淀和转移细胞到一个新的T - 75或T - 175烧瓶中含有25毫升（T - 75）或50毫升（T - 175）的RPMI 1640含10％胎牛血清，1％青霉素/链霉素和20 ng / mL的人IL - 2或IL - 15。 </li><li>长为4-7天的淋巴细胞。如果与一个T - 75烧瓶开始，文化需要扩大和转移到了T - 175烧瓶后1-2天。 </li></ol> （3） 在体外淋巴细胞迁移实验<ol><li>迁移实验前1天，外套一个玻璃底的0.17毫米与20μg/ mL的蛋白A或摹在PBS过夜4盘° C。 </li><li>广泛的菜洗净，用PBS。 </li><li>固定人类ICAM-1/Fc（10微克/毫升）和人力SDF - 1的PBS液中加入这些试剂的菜，并在室温下孵育4个小时（2微克/毫升）。 </li><li>准备首先确定的文化中的T - 175使用一个hemacytometer烧瓶细胞密度T淋巴细胞。应使用约2-5 × 10 5细胞，每道菜，实现了细胞密度适当的迁移分析。 </li><li>两次用PBS洗T淋巴细胞，然后悬浮在1毫升含有1毫克/毫升D -葡萄糖的L - 15媒体。 </li><li>涂有ICAM-1/Fc和SDF - 1广泛用PBS洗净的菜。 </li><li> T淋巴细胞在1毫升媒体传输的菜，和他们保持在37 ° C。 </li></ol> 4。使用NIS Elements软件捕获图像序列<ol><li>打开NIS Elements软件。 </li><li>在相机设置菜单中，选择“2x2的分级”，实时成像和图像捕捉模式。 </li><li>转到“应用程序”菜单中选择定义/运行实验。 </li><li>选择图像和图像采集序列的总长度之间的时间长度。 </li><li>按“运行”按钮开始图像采集。 </li><li>细胞可以跟踪和迁移参数（速度，路径长度，位移等）的量化使用几个软件包，包括ImageJ，AutoQuant或Volocity（图1）。 </li><li>要生成一个“蜘蛛网情节，”XY坐标，为每一个细胞和时间点收集（图2），并投射到一个共同的出发点，在每个细胞的起源（图3）图。 </li></ol> 5。代表性的成果文化6天或IL - 2或IL - 15，T淋巴细胞的存在&gt; 98％的细胞，积极CD3 +染色，以及积极的CD4 +和/或CD8染色确定。对于IL - 2，我们发现83％的CD4 +细胞，15％CD8 +和&lt;1％的CD4 + CD8 +细胞。对于IL - 15，我们发现88％的CD4 +细胞，11％，CD8 +和&lt;1％的CD4 + CD8 +细胞。细胞在T淋巴细胞迁移ICAM-1/SDF-1基板上，展出了约15微米/分钟，可超过1个小时的时间内持续的速度。 ICAM-1/SDF-1 T对淋巴细胞迁移依赖于LFA - 1介导的粘附，作为一种抗LFA - 1配体阻断抗体严重抑制迁移。 <img src="/files/ftp_upload/2017/2017fig1.jpg" alt="图1" /> 图1。细胞跟踪从全血中分离，并在6天的IL - 2或IL - 15培养的T淋巴细胞，T淋巴细胞的迁移。坚持和迁移10μg/ mL的ICAM - 1和镀膜玻璃底菜2μg/ mL的SDF - 1。图片被收购30分钟，每10秒。每幅图像的细胞进行鉴定和跟踪时间超过使用Volocity软件。这部影片展示了T淋巴细胞〜15微米/分钟的速度的随机迁移。 <a href="http://www.jove.com/files/ftp_upload/2017/2017_Kim_Movie1.mov">点击这里看视频。</a> 电影1。细胞T淋巴细胞的迁移跟踪。从全血中分离和培养中存在的的T淋巴细胞IL - 2或6天的IL - 15是坚持和迁移涂有10微克/毫升ICAM - 1和2微克/毫升SDF - 1的玻璃底船菜。图片被收购30分钟，每10秒。每幅图像的细胞进行鉴定和跟踪时间超过使用Volocity软件。这部影片展示了T淋巴细胞〜15微米/分钟的速度的随机迁移。 <img src="/files/ftp_upload/2017/2017fig2.jpg" alt="图2" /> 图2。时空单元格的位置。每次使用Volocity软件获得的每一个细胞的XY坐标。 <img src="/files/ftp_upload/2017/2017fig3.jpg" alt="图3" /> 图3。“蛛网的阴谋。”使用XYt每一个细胞，随机选择了15个细胞，并绘制一个共同的起点在原点的坐标。蛛网图相比较，给出了一个移民之间的差异不同实验条件下的快速可视化的描述。图解T淋巴细胞迁移控制的条件下（左图）和抗LFA - 1的配体阻断抗体（右图）的存在。

<ol>
	<li>Hyun, Y. M., Chung, H. L., McGrath, J. L., Waugh, R. E., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+integrin+VLA-4+localizes+to+the+lamellipodia+and+mediates+t+cell+migration+on+VCAM-1.">Activated integrin VLA-4 localizes to the lamellipodia and mediates t cell migration on VCAM-1.</a> J Immunol. 183, 359-369 (2009).</li><li>Morin, N. A., Oakes, P. W., Hyun, Y. M., Lee, D., Chin, Y. E., King, M. R., Springer, T. A., Shimaoka, M., Tang, J. X., Reichner, J. S., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonmuscle+myosin+heavy+chain+IIA+mediates+integrin+LFA-1+de-adhesion+during+T+lymphocyte+migration.">Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration.</a> The Journal of experimental medicine. 205, 195-205 (2008).</li><li>Hyun, Y. M., Lefort, C. T., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+integrins+and+their+ligand+interactions.">Leukocyte integrins and their ligand interactions.</a> Immunologic research. , (2009).</li></ol>

在这个实验中，我们提供了一个简单的系统来分析初级人类T淋巴细胞的活力细节。体外迁移实验已用于解剖的许多分子和参与各种细胞类型的运动的信号转导通路的作用。要记住，从我们的协议设计自己的实验时，一些关键的控制包括：1）非粘性或不整合胶粘剂基材，如牛血清白蛋白（BSA）或聚左旋赖氨酸（PLL），涂料，分别为2 ）整合特定的封闭抗体治疗，无刺激信号，如SDF - 1在我们?...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>RPMI 1640</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11875</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH300070.03</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>1-Step Polymorphs</td>
<td>&nbsp;</td>
<td>Accurate Chemical</td>
<td>AN221725</td>
<td>&nbsp;</td>
<tr>
<td>PHA</td>
<td>&nbsp;</td>
<td>Remel/Fisher Scientific</td>
<td>R30852801</td>
<td>&nbsp;</td>
<tr>
<td>Rec. human IL-2</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>202-IL</td>
<td>&nbsp;</td>
<tr>
<td>Rec. human IL-15</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>247-IL</td>
<td>&nbsp;</td>
<tr>
<td>Protein G</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>19459</td>
<td>&nbsp;</td>
<tr>
<td>Rec. human ICAM-1/Fc</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>720-IC</td>
<td>&nbsp;</td>
<tr>
<td>Rec. human SDF-1</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>350-NS</td>
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human t lymphocyte isolation, culture and analysis of migration in vitro

T淋巴细胞的迁移涉及到与其他细胞或细胞外基质蛋白表达的配体的细胞表面整合的粘合剂的相互作用。从低亲和力状态激活在细胞前沿的高亲和力状态的精确时空的整合是很重要的T淋巴细胞迁移<sup&gt; 1</sup&gt;。同样，细胞后缘内陷，或uropod，是一个必要的步骤，保持持久的整合依赖的T淋巴细胞的活力<sup&gt; 2</sup&gt;。许多治疗方法，自身免疫性或炎症性疾病的目标整合为手段来抑制白细胞的过度招聘和迁移<sup&gt; 3</sup&gt;。要研究的分子事件，调节人体的T淋巴细胞迁移，我们利用<em&gt;在体外</em&gt;系统来分析一个两维的基板，模仿，T淋巴细胞从血管招聘过程中遇到的环境的细胞迁移。 T淋巴细胞是首次分离出器官捐献者，然后激发和培养七到十天。在实验中，T淋巴细胞是允许涂上间粘附分子1（ICAM - 1），LFA - 1整合的配体，基质细胞衍生因子-1（SDF - 1）基板上坚持和迁移。我们的数据表明，T淋巴细胞表现出〜15微米/分钟的迁徙速度。可以抑制T淋巴细胞的迁移整合封锁<sup&gt; 1</sup&gt;或抑制细胞的肌动球蛋白机械，调节细胞迁移<sup&gt; 2</sup&gt;。

Watch this Scientific Journal Video about Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro at JoVE.com

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

T淋巴细胞的迁移过程中出现归巢淋巴器官，从血管中退出，进入周围组织。在这里，我们描述了一个可以用来分析T淋巴细胞迁移的协议<em&gt;在体外</em&gt;。

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 ...

human-t-lymphocyte-isolation-culture-analysis-migration

Research

JoVE Journal

Immunology and Infection

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

46.1K 次观看.  University of Rochester. T淋巴细胞的迁移过程中出现归巢淋巴器官，从血管中退出，进入周围组织。在这里，我们描述了一个可以用来分析T淋巴细胞迁移的协议在体外。

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

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

科研

免疫与感染

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

成像的CD4 T细胞间质迁移发炎真皮

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

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 Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

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 ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

人单核细胞趋化体外和小鼠淋巴细胞贩运体内

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic 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 ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

小鼠边缘区 b 细胞剪切流诱导迁移的体外分析

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...