Name: radial mobility and cytotoxic function of retroviral replicating vector transduced, non-adherent alloresponsive t lymphocytes
Uploaded: 2015-02-11T13:00:00
Description: Watch this Scientific Journal Video about Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes at JoVE.com

支持由NIH R01 CA121258，R01 CA125244，R01 CA154256，NIH / NCATS UCLA CTSI授权号UL1TR000124，USAMRMC W81XWH-08-1-0734，和琼·S·霍姆斯纪念研究基金的一部分。 MJH和GCO接到琼S.福尔摩斯纪念博士后奖学金，在加州大学洛杉矶分校的支持。 CSM的装置是由创新科学方法获得：www.creative-sci.com。慢病毒载体是从加州大学洛杉矶分校矢量的核心，这是支持CURE / P30 DK041301收到。

1.将准备<ol><li>将细胞沉淀歧管滑入杀菌袋和密封用高压灭菌胶带。面对滑板袋的纸侧，以避免在井中塑料沉积物的特氟隆涂层的一面。 </li><li>高压釜袋15分钟，在121℃。 </li><li>在无菌的150×15毫米的无菌培养皿中取出的幻灯片从生物安全柜和地点内杀菌袋。多达四个幻灯片每盘可以容纳。广场旁边的幻灯片35×10毫米的培养皿。加入2-3毫升无菌H 2澳提供加湿。 </li><li>预涂载玻片用聚-D-赖?...

<ol>
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肿瘤细胞中的单个细胞悬浮液移液到特氟隆掩模滑动的孔中。使细胞附着，然后在湿润的5％CO 2，37℃培养箱（ 图1A），形成单分子膜。从单层衍生既定单层或ECM蛋白可收获这些测定（ 图1B）。标记有重要的荧光染料或转导的载体编码的EMD效应T淋巴细胞接种到使用CSM阱的中央。然后非粘附效应T淋巴细胞的迁移上的单层或ECM蛋白随时间的能力进行了评估和量化的光?...

径向单层细胞迁移测定法最初开发到载玻片上涂覆有细胞外基质（ECM）蛋白5-7或具有单个ECM组分，如纤连蛋白或层粘连蛋白1,2-测量粘附的肿瘤细胞1-4的浸润特性。该技术涉及接种肿瘤细胞的单细胞悬浮液在使用不锈钢细胞沉降支管（CSM）的孔的中心。沉淀后，将肿瘤细胞将附着在井和在初始细胞群随时间的直径的改变的底物用于建立横向运动的速率。径向单层细胞迁移测定法提供了一种视觉优势所采用的transwell板，以测定细胞的体外迁徙功能等现有的方法;这些测定法是非利于成像8。同时，它也是在选择时间点所提供的自由量很大■当迁移评估，对时间点的数量没有限制的研究员能沉淀后，选择图像。 因为迁移的能力是对非贴壁细胞的一个重要的功能，特别是在免疫治疗或者它们可以用作递送载体为病毒载体领域，我们适于使用CSM来评价非粘附细胞的迁移类型对肿瘤细胞单层，除了ECM蛋白。的显微镜可视化上存活的肿瘤细胞单层非粘附细胞的迁移，在复杂的ECM从肿瘤，或单个ECM组分分离的额外的好处，使该测定的通用性。雇用孔涂有单一胞外蛋白分析不准确地反映ECM组织基质或肿瘤的细胞将通过迁移体内 。 这里，我们使用同种异体细胞毒性T淋巴细胞（alloCTL），敏感地认识到主要histocomp使用单向混合淋巴细胞肿瘤细胞反应（MLTR）或混合淋巴细胞反应（MLR）9，作为我们的代表性非粘附细胞类型atibility复合体（MHC）蛋白。我们测试了人和鼠源的细胞。当迁移测定肿瘤单层，采用的肿瘤细胞或者部分相关指标，显示一些对用于敏感的效应，还是完全相关的目标，拥有全套MHC分子的细胞群发现了同样的MHC蛋白质的效应已被致敏对。在一些实验中，我们使用了荧光CellTracker红色CMPTX或细胞增殖染料eFluor 670效应细胞和靶细胞之间进行区分。我们还使用转用病毒载体编码荧光蛋白作为可视化细胞的另一种方式。对于某些分析，我们转了alloCTL与逆转录病毒的复制载体（RRV）编码翡翠绿（EMD）荧光蛋白10,11;对于超视距器，肿瘤细胞被转导的慢病毒载体编码mStrawberry。 所述alloCTL被通过歧管的一个通道进入任一肿瘤细胞单层或ECM从肿瘤细胞单层收获的中心接种。粘附和非粘附细胞相互作用显现由光和/或通过在一段时间的荧光显微镜。中断在肿瘤细胞单层在低功率，或肿瘤细胞具有片段化的细胞核在高功率是细胞损伤的由裂解和细胞凋亡的指标，分别。我们创建数字表面强度荧光地图显示非粘附荧光T细胞在单层培养的迁移。我们还注意到毒性集群的形成叠加的非贴壁alloCTL之后主义及其对贴壁胶质瘤细胞单层。同时，观察RRV-EMD从alloCTL到胶质瘤单层水平传导。

<table border="0" cellpadding="0" cellspacing="0" width="920">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Name of Reagent/ Equipment</td>
			<td style="width:203px;">Company</td>
			<td style="width:239px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:312px;">Teflon-masked microscope slides</td>
			<td style="width:203px;">Creative Scientific Methods</td>
			<td style="width:239px;">CSM002</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:312px;">Cell Sedimentation Manifold</td>
			<td style="width:203px;">Creative Scientific Methods</td>
			<td style="width:239px;">CSM001</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Petri Dish, 150mm</td>
			<td style="width:203px;">Corning</td>
			<td style="width:239px;">430597</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Petri Dish, 35mm</td>
			<td style="width:203px;">Corning</td>
			<td style="width:239px;">430588</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Phosphate-Buffered Saline (PBS)</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">21-040</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">20 &#956;L Pipetman</td>
			<td style="width:203px;">Gilson</td>
			<td style="width:239px;">F123600</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">200 &#956;L Pipetman</td>
			<td style="width:203px;">Gilson</td>
			<td style="width:239px;">F123601</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">200 &#956;L pipette tips</td>
			<td style="width:203px;">VWR</td>
			<td style="width:239px;">89003-060</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Distilled, deionized water (sterile)</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">25-055</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Trypsin</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">25-054-CL</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Celltracker Red CMPTX</td>
			<td style="width:203px;">Invitrogen</td>
			<td style="width:239px;">C34552</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">TrypLE Express (optional)</td>
			<td style="width:203px;">Gibco</td>
			<td style="width:239px;">12604</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Tumor cell culture media (e.g. DMEM)</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">10-013</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">AIM-V serum-free media</td>
			<td style="width:203px;">Invitrogen</td>
			<td style="width:239px;">12055-083</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Fetal Bovine Serum</td>
			<td style="width:203px;">Omega Scientific</td>
			<td style="width:239px;">FB-02</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Inverted microscope</td>
			<td style="width:203px;"></td>
			<td style="width:239px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">SPOT Advanced</td>
			<td style="width:203px;">Diagnostic Instruments</td>
			<td style="width:239px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Poly-D-Lysine</td>
			<td style="width:203px;">Millipore</td>
			<td style="width:239px;">A-003-E</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Fibronectin</td>
			<td style="width:203px;">BD</td>
			<td style="width:239px;">354008</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:312px;">31/2 x 51/4 Autoclave Pouches</td>
			<td style="width:203px;">Crosstex</td>
			<td style="width:239px;">SCXS2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Trypan Blue</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">25-900-CI</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Cell Proliferation eFluor 670</td>
			<td style="width:203px;">eBioscience</td>
			<td style="width:239px;">65-0840-85</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">ImageJ</td>
			<td style="width:203px;">NIH</td>
			<td style="width:239px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

radial mobility and cytotoxic function of retroviral replicating vector transduced, non-adherent alloresponsive t lymphocytes

我们报告的径向单层细胞迁移测定法的一个新颖的适应，首次报道以测量粘附的肿瘤细胞的细胞外基质蛋白的径向迁移，用于测量荧光标记，非粘附人或鼠效应免疫细胞的运动性。这种技术采用一个不锈钢歧管和10以及特氟隆滑动灶性沉积非贴壁的T细胞变成制备与任一汇合的肿瘤细胞单层或细胞外基质蛋白的孔中。光和/或多信道荧光显微镜被用于随时间跟踪效应细胞的移动和行为。荧光染料和/或荧光的转基因编码用于差异标记的细胞类型进行成像的病毒载体。这种方法是从类似型的体外测定该跟踪水平或垂直的迁移/侵袭利用滑动室，琼脂或Transwell小板明显。该法允许详细的影像数据，以BË与特定的荧光标记区分不同类型的细胞收集;即使特定的细胞亚群（ 即 ，转导/ nontransduced）可以被监控。使用对应于所述转移的小区类型特定的荧光通道中产生的表面强度的荧光曲线。这允许在特定时间更好的可视化的非粘附免疫细胞迁移的。因此能够收集的其他效应细胞的功能，如细胞毒性或从效应器病毒载体的转移证据到靶细胞，以及。因此，该方法允许研究人员用显微镜记录差异标记的，非粘附的细胞 - 细胞间的相互作用与各种类型的贴壁细胞。这样的信息可以是在生物-操纵或激活免疫细胞类型，其中的功能性的视觉证据期望与肿瘤靶细胞将其用于癌症治疗前的评估特别相关。

对于荧光蛋白编码病毒载体可以除了，或代替，荧光染料可以使用。病毒转导应提前运动检测来进行。既粘附和非粘附的细胞类型可以是差异标记。的协议转导将取决于载体的使用的类型。在这里，我们转导在图3，图5和图6与RRV-EMD至少两天的alloCTL提前使用协议测定的描述12。我们还使用的慢病毒载体，CMV-草莓-IRES-FLUC2，转导的肿瘤细胞以表达mStrawberry红色荧光蛋?...

Watch this Scientific Journal Video about Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes at JoVE.com

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

径向移动性和逆转录病毒复制的载体转导，非粘附Alloresponsive T细胞的杀伤功能

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...

Research

JoVE Journal

Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

扩大目标，脐血细胞毒性T淋巴细胞，巨细胞病毒，EB病毒，腺病毒

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

定向分化诱导性多潜能干细胞向T淋巴细胞

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

静电粘附实验对整合素激活的T细胞研究

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

从人类腭扁桃体B和T淋巴细胞有效隔离协议

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

骨髓祖细胞的逆转录病毒转导到生成T细胞受体Retrogenic小鼠

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

发电<em&gt;德诺</em&gt;抗原特异性人T细胞受体通过中心的Hemichain的逆转录病毒转导

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...