Name: human t lymphocyte isolation, culture and analysis of migration in vitro
Uploaded: 2010-06-01T17:00:00
Description: Watch this Scientific Journal Video about Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro at JoVE.com

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?...

<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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<td>RPMI 1640</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11875</td>
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<td>FBS</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH300070.03</td>
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<td>Penicillin-Streptomycin</td>
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<td>Gibco</td>
<td>15140</td>
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<td>1-Step Polymorphs</td>
<td>&nbsp;</td>
<td>Accurate Chemical</td>
<td>AN221725</td>
<td>&nbsp;</td>
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<td>PHA</td>
<td>&nbsp;</td>
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<td>Rec. human IL-2</td>
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<td>R&amp;D Systems</td>
<td>247-IL</td>
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<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>19459</td>
<td>&nbsp;</td>
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<td>Rec. human ICAM-1/Fc</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>720-IC</td>
<td>&nbsp;</td>
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<td>Rec. human SDF-1</td>
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<td>R&amp;D Systems</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 ...

Research

JoVE Journal

Immunology and Infection

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

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

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

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

Mouse NaÃƒÆ’Ã‚Â¯ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

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

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

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

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

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

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

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

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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

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, ...