Name: rapid and robust analysis of cellular and molecular polarization induced by chemokine signaling
Uploaded: 2014-12-12T14:00:00
Description: Watch this Scientific Journal Video about Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling at JoVE.com

笔者极大地承认皮埃尔Bourdoncle，托马斯·吉尔伯特和科钦成像设备的路易丝Rimbault。这项工作是由INSERM，法国国家科学研究中心和法甲国立驳乐癌症（队报labellisée）的支持。

1.准备T淋巴细胞<ol><li>准备主鼠标或人类T细胞所描述10,11。 </li><li>培养的人T细胞在完全的RPMI培养基中，用10％的人血清AB以2的密度- 3×10 6个细胞/ ml。 注意：使用胎牛血清（FCS）代替人血清的能引起人T细胞的培养的大部分的自发极化。在此期间5天，进一步处理他们在任何时候 - 保持这些细胞的培养4。 </li><li>保持小鼠的T细胞在一个相似的细胞密度在补充有10％FCS的完全RPMI?...

<ol>
	<li>Rossi, D., Zlotnik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+chemokines+and+their+receptors.">The biology of chemokines and their receptors.</a> Annu Rev Immunol. 18, 217-242 (2000).</li><li>Thelen, M., Stein, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+chemokines+invite+leukocytes+to+dance.">How chemokines invite leukocytes to dance.</a> Nat Immunol. 9 (9), 953-959 (2008).</li><li>Rougerie, P., Rho Delon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTPases:+masters+of+T+lymphocyte+migration+and+activation.">GTPases: masters of T lymphocyte migration and activation.</a> Immunol Lett. 142 (1-2), 1-13 (2012).</li><li>Sanchez-Madrid, F., del Pozo, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+polarization+in+cell+migration+and+immune+interactions.">Leukocyte polarization in cell migration and immune interactions.</a> Embo J. 18 (3), 501-511 (1999).</li><li>Adams, D. 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=Hepatocyte+growth+factor+and+macrophage+inflammatory+protein+1+beta:+structurally+distinct+cytokines+that+induce+rapid+cytoskeletal+changes+and+subset-preferential+migration+in+T+cells.">Hepatocyte growth factor and macrophage inflammatory protein 1 beta: structurally distinct cytokines that induce rapid cytoskeletal changes and subset-preferential migration in T cells.</a> Proc Natl Acad Sci U S A. 91 (15), 7144-7148 (1994).</li><li>Rougerie, P., 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=Fam65b+is+a+new+transcriptional+target+of+FOXO1+that+regulates+RhoA+signaling+for+T+lymphocyte+migration.">Fam65b is a new transcriptional target of FOXO1 that regulates RhoA signaling for T lymphocyte migration.</a> J Immunol. 190 (2), 748-7455 (2013).</li><li>Real, E., Faure, S., Donnadieu, E., Delon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+Atypical+PKCs+regulate+T+lymphocyte+polarity+and+scanning+behavior.">Cutting edge: Atypical PKCs regulate T lymphocyte polarity and scanning behavior.</a> J Immunol. 179 (9), 5649-5652 (2007).</li><li>Negulescu, P. A., Krasieva, T. B., Khan, A., Kerschbaum, H. H., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarity+of+T+cell+shape,+motility,+and+sensitivity+to+antigen.">Polarity of T cell shape, motility, and sensitivity to antigen.</a> Immunity. 4 (5), 421-430 (1996).</li><li>Zuba-Surma, E. K., Kucia, M., Abdel-Latif, A., Lillard, J. W., Ratajczak, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ImageStream+System:+a+key+step+to+a+new+era+in+imaging.">The ImageStream System: a key step to a new era in imaging.</a> Folia Histochem Cytobiol. 45 (4), 279-290 (2007).</li><li>Matheu, M. P., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+CD4++T+cells+from+mouse+lymph+nodes+using+Miltenyi+MACS+purification.">Isolation of CD4+ T cells from mouse lymph nodes using Miltenyi MACS purification.</a> J Vis Exp. (9), 409 (2007).</li><li>James, E. A., LaFond, R., Durinovic-Bello, I., Kwok, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+antigen+specific+CD4++T+cells+using+MHC+class+II+tetramers.">Visualizing antigen specific CD4+ T cells using MHC class II tetramers.</a> J Vis Exp. (25), (2009).</li><li>Wabnitz, G. 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=L-plastin+phosphorylation:+a+novel+target+for+the+immunosuppressive+drug+dexamethasone+in+primary+human+T+cells.">L-plastin phosphorylation: a novel target for the immunosuppressive drug dexamethasone in primary human T cells.</a> Eur J Immunol. 41 (11), 3157-3169 (2011).</li><li>Freeley, M., 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=L-plastin+regulates+polarization+and+migration+in+chemokine-stimulated+human+T+lymphocytes.">L-plastin regulates polarization and migration in chemokine-stimulated human T lymphocytes.</a> J Immunol. 188 (12), 6357-6370 (2012).</li></ol>

使用最近的成像流技术术，快速和翔实门控策略进行分析诱导的趋化因子刺激的细胞和分子事件呈现。从一个单一的实验，可以得到的信息的两个主要类型：诱导的趋化因子刺激和不同的蛋白质的过程中的极化过程的亚细胞分布的细胞形态的变化。有趣的是，证据还提供了用于分析大量细胞的，这允许统计鲁棒性和整个细胞群体的一小部分相关的分析的可能性。据报道，这种技术也可用于在免疫?...

趋化因子是吸引细胞专门的地点1小的可溶性蛋白质。因此，它们参与细胞在组织中的正确定位，在发展和生理的一个关键功能。免疫系统是无例外，因为它依赖于在音乐会其作用是进行有效的免疫应答的许多不同类型的细胞的作用。通过控制在一个给定的状态下的一种免疫细胞类型的具体位置，趋化因子是预先需要外来抗原可被检测并中和之前。 在T淋巴细胞特别是趋化因子绑定到特定的表面受体其中，在接合时，引起许多细胞内的信号（钙上升，ERK磷酸化，卢GTP酶的活化，增加整合的亲和力和细胞骨架的改变）有利于T细胞的活力2,3。在细胞水平，人们可以观察到形态学改变时的趋化因子刺激引起。这些变化在细胞形状是在T细胞中特别引人注目：静息T细胞具有珠状轮的形态在血流中行进时。然而，在炎症部位或淋巴器官的附近的趋化因子的存在的感测会改变T细胞的形状，现在采用一种典型的“手镜”的形态组成的双极形式：前缘在前面和后缘，或尾肢，在后面4。此外，细胞内的组分可以分离成偏振性T细胞的这两个相对的区域，以维持迁移。例如，在趋化因子刺激5和聚合的肌动蛋白微丝聚合增加积聚在一个极化T细胞2的前面。另一方面，一些蛋白质，如Ezrin蛋白 - 根蛋白 - 膜突（ERM）家族，质膜链接到皮质F-肌动蛋白细胞骨架，磷酸化的蛋白质重新定位于极化的尾肢T细胞6。有趣的是，我们和其他人已经表明，所需的T细胞迁移该偏振过程。事实上，与极化干扰任何治疗会抑制细胞运动。例如，抑制非典型蛋白成员的活性的激酶C（PKC）家族，蛋白激酶Cζ和PKCι块T细胞极化和树突细胞7的其迁徙扫描过程。 T细胞极化也受卢GTP酶。我们已经表明，RhoA活性的由最近描述Fam65b蛋白调制与形态学T细胞的变化和它们的能力干涉以在Transwell小试验6迁移。作为极化是细胞运动的前提步骤，它是这样的关键是能够量化它作为趋化因子应答的主读出。细胞形态改变，以前手工测量8。然而，这种量化的非常费时，因此通常只有几几十细胞都考虑在内。 在这里，一个新的方法，提出了快速量化暴露于趋化因子刺激的T淋巴细胞的形状的改变的程度。流式细胞技术的成像流（ 见表特定试剂/设备 ）的情况下，它结合了流式细胞仪和显微镜9的优势，有效地量化的趋化因子刺激的不同条件极化的细胞的量。除了形态学改变，人们可以使用该技术鲁棒测量的定量，还能够评价的变化时的趋化因子信号传导的一些蛋白质的亚细胞定位。

<table border="0" cellpadding="0" cellspacing="0" width="569">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Name of the material/Equipment</td>
			<td style="width:95px;">Company</td>
			<td style="width:80px;">Catalog number</td>
			<td style="width:255px;">Comments/Description (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">RPMI</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">61870-010</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Human serum AB</td>
			<td style="width:95px;">PAA</td>
			<td style="width:80px;">C11-021</td>
			<td style="width:255px;">Pre-heat to inactivate the complement.</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:140px;">Fetal calf serum</td>
			<td style="width:95px;">PAN Biotech</td>
			<td style="width:80px;">P30-3300</td>
			<td style="width:255px;">Pre-heat to inactivate the complement.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:140px;">Human T cell nucleofactor kit</td>
			<td style="width:95px;">Lonza</td>
			<td style="width:80px;">VCA-1002</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Murine IL7</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">217-17</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:140px;">HBSS</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">14025-050</td>
			<td style="width:255px;">Warm in 37 &#176;C water bath before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Hepes</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">15630-056</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:140px;">Murine CCL19</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">250-27B</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:140px;">Human CCL19</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">300-29B</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells.</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:88px;width:140px;">Human CXCL12</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">300-28A</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells. This chemokine can also be used on mouse T cells.</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:140px;">PFA</td>
			<td style="width:95px;">Electron Microscopy Sciences</td>
			<td style="width:80px;">157-8-100</td>
			<td style="width:255px;">This is a 8 % PFA solution in water. Mix volume to volume with 2X PBS to obtain a 4 % PFA solution in PBS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">&#160;BSA</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:80px;">A3059</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Saponin</td>
			<td style="width:95px;">Fluka</td>
			<td style="width:80px;">84510</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">Alexa Fluor 594 phalloidin</td>
			<td style="width:95px;">Invitrogen</td>
			<td style="width:80px;">A12381</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">FITC-anti-HLA-ABC antibody</td>
			<td style="width:95px;">Beckman Coulter</td>
			<td style="width:80px;">IM1838</td>
			<td style="width:255px;">clone B9.12.1</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">Anti-P-ERM antibody</td>
			<td style="width:95px;">Cell Signaling Technology</td>
			<td style="width:80px;">3149P</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:140px;">ImageStreamx Mark II</td>
			<td style="width:95px;">Amnis</td>
			<td style="width:80px;"></td>
			<td style="width:255px;"></td>
		</tr>
	</tbody>
</table>

rapid and robust analysis of cellular and molecular polarization induced by chemokine signaling

细胞响应由失去其圆形形状在这个过程被称为极化，并通过改变许多蛋白质​​的亚细胞定位趋化因子刺激。经典的成像技术已被用来研究这些现象。然而，他们所需要的手工采集许多细胞后跟形态和共定位几十细胞染色的耗时定量。这里，快速和有效的方法被描述为在由几千利用流式细胞技术的成像流动，结合了显微镜的优点与这些细胞仪的细胞的样品研究了这些现象。使用与CCL19和染色MHC I类分子和丝状肌动蛋白刺激的T淋巴细胞，一个选通策略提出同时测量形状的改变的程度和标记物的共定位该受CCL19信令的程度。此外，这门控策略使我们能够observë丝状肌动蛋白（正面）和磷酸化Ezrin蛋白 - 根蛋白 - 膜突（磷ERM）的蛋白质（在后面）中的CXCL12刺激后偏振性T细胞的分离。这种技术也是有益的，观察偏振的两种不同元素的阻挡效果：由PAR6 /非典型PKC信号传导途径的突变体的药理抑制剂和表达抑制肌动蛋白聚合的。因此，有证据表明，这种技术是分析这两种形态的改变和蛋白质再分配有用。

这里选择的第一个例子是关于使用与CCL19刺激的人初级T淋巴细胞的。然而，同样的策略可用于原发性小鼠T细胞，T细胞系或任何其他细胞类型响应于趋化因子刺激。这里介绍的门控战略包括一系列选择聚焦的事件，那么单个细胞窗口。最后荧光强度的范围被这里作为一个例子，随后在两个标记：对MHC I类HLA-ABC和丝状肌动蛋白为静息（ 图1）或CCL19刺激（ 图2）的T淋巴细胞。...

Watch this Scientific Journal Video about Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling at JoVE.com

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

细胞与分子极化的快速和强大的分析趋化因子诱导信号

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many ...

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Immunology and Infection

Unit 1

Cell Polarization and Migration

SCIENTISTS IN ACTION

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &#945;-actinin by Confocal Imaging

胞内相互作用的可视化和量化脑膜炎奈瑟菌和人类α-辅肌动蛋白共聚焦成像

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

科研

免疫与感染

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

滴流和旋转盘反应器使用金黄色葡萄球菌生物膜分析

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

艾滋病毒的成像1信封引起的病毒学突触信号合成脂双层

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

一个实时，同步成像的TCR和其相关的信号转导蛋白的T​​IRF显微镜技术

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

上皮损伤制作分析<em&gt;阿米巴</em&gt;感染

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

在小鼠B淋巴细胞染色体畸变所PNA-FISH快速分析

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

快速分子检测和流感病毒A和B的分化

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

T淋巴细胞有丝分裂原诱导芽生的快速定量辨识免疫调节药物

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

分子互补亲和纯化 (BiCAP) 对多蛋白信号络合物的解剖

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

登革热病毒诱发急性病毒性脑炎样疾病的鼠害模型

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is ...