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培养基。在10毫微克/毫升IL7存在立即使用或隔天，后O / N培养在37℃。 </li><li>培养CEM人T-细胞系在完全RPMI补加10％FCS中。 </li></ol> 2.趋化因子刺激<ol><li>在补充了10mM的HEPE温暖的HBSS培养基洗每个实验条件5×10 5个细胞秒。对于一些实验中，前一天共转染原代人T细胞与2微克pmaxGFP和8微克的pcDNA3.1 +（空载体对照），蛋白激酶Cζ激酶死的，或N末端​​PAR6质粒。 </li><li>悬浮在温暖的HBSS-HEPES中的细胞沉淀（用实验条件0.3毫升X号）。 </li><li>分发在1.5ml微量离心管中的细胞。 注:因此，对应于一个单一的实验条件1管将含有5×10 5个细胞在0.3毫升的HBSS-HEPES培养基。对于一些实验中，加入500纳米Latrunculin A或车辆（DMSO）孵育细胞趋化因子刺激前30分钟。 </li><li>添加10〜500毫微克/毫升的CCL19或CXCL12趋化因子中的每个管中。 注:我们通常使用10 - 200毫微克/毫升的趋化因子对人类T细胞。 CEM细胞不表达CCR7受体结合CCL19。因此，CEM细胞将仅能够以一个CXCL12刺激作出响应。此外，使用较高浓度的趋化因子（300 - 500 N克/毫升）极化小鼠T细胞。 </li><li>翻转管几次，分别孵育它们在37℃的水浴中8或10分钟为主要的T细胞或CEM。 </li><li>通过加入到每个管0.3ml的含有2％多聚甲醛（PFA）和10mM的Hepes在PBS中的热溶液停止该极化过程。翻转试管孵育它们在37℃下5分钟。 </li></ol> 3.染色<ol><li>从离心管转移至细胞流式细胞管中。 </li><li>完全填充管与含有1％牛血清白蛋白（BSA），0.5毫摩尔EDTA及10毫摩尔的Hepes的PBS室温溶液中。 </li><li>离心管中，在460 XG 4分钟。 </li><li>弃去上清并将细胞重新悬浮在100μl的含5％FCS的PBS室温溶液中。 </li><li>添加5微升的FITC的抗HLA-ABC在每个管中，涡流它们，它们孵育30分钟，在室温，避光。 </li><li>用PBS含5％FCS洗涤一次细胞，一ND一次PBS而已。 </li><li>在RT:加入500μl1％PFA中，孵育10分钟，然后用含有1％BSA，0.5毫摩尔EDTA和10mM HEPES的PBS溶液洗涤细胞。 </li><li>弃去上清液，完全填充管以含0.2％BSA，0.1％皂甙，0.5毫摩尔EDTA和10mM的Hepes（透化缓冲液）的PBS溶液。 </li><li>在该培养基中洗涤细胞两次。 </li><li>翻转管以除去介质，涡旋它们以解离细胞团。 </li><li>添加0.25微克/毫升TRITC-鬼笔环肽和/或1:100稀释的抗P-ERM抗体与各管中，涡流它们并温育30分钟，在室温。 </li><li>与通透缓冲洗涤细胞。孵育如有必要，荧光第二抗体。 </li><li>悬浮细胞在200微升PBS，并将其转移到离心管。 注:细胞准备收购。另外，同时保持每管200微升最大总容积，加浓PFA到最终1％为了集中保存染色，如果细胞不被使用的同一天，以便进一步处理。 </li></ol> 4.图像采集与分析<ol><li>开关上的装置（ 见表特定试剂/设备 ），并让它根据制造商的说明校准自身。 注:INSPIRE软件有40X的目标（0.75 NA）使用。思想3.0软件被用于所有报告的量化值。 </li><li>制备了一系列的采集窗口，并且可以保存在一个模板以供将来的实验。得出量化的RMS梯度值直方图。从"聚焦"人群中，对RMS梯度直方图选择，delimitate的"单细胞"群对区域的直方图。一旦离心管已插入在机器中，调整激光功率，对单个细胞栅极和获得5000 T淋巴细胞。 </li><li>在软理念洁具，开收购文件，并创建为每个单元获得的直方图，将显示为亮场图像梯度RMS值的值（通道1）。借鉴RMS梯度直方图认为细胞表现出RMS上述57梯度值门。 注:RMS梯度允许考虑到在分析仅T淋巴细胞是在焦平面。我们已经凭经验确定，表现出的R​​MS梯度值优于57个体细胞是在焦平面，并且可以精确地考虑。 </li><li>在分析/面具菜单，创建一个新的面具，被称为从在明图像M01，侵蚀了2个像素的形态面具侵蚀（M01,2）。然后，在分析/功能菜单，创建面积，计算出的侵蚀（M01,2）面具的一项新功能。从在前面的门中选择的细胞中，打开一个直方图表示使用这个新创建的掩模单元的面积。 注:该形态口罩可用通过默认比小区的实际尺寸要大得多。因此，更精确的侵蚀它最多2个像素。 </li><li>对个人的T细胞画门，不包括校准珠和碎片（小面积）和双峰（大区）。在每次采集调整这个门。 注:在细胞大小的变化会影响到门的位置。小鼠T细胞是比人类T细胞它们本身比CEM细胞小较小，每种细胞类型的区域将正比于它的大小。 </li><li>考虑到单，在焦细胞进行门控，在前面的步骤中，打开一个点图构成的原料最大象素上的HLA-ABC染色（通道2中，x轴）作为原料最大象素上的鬼笔环肽的功能染色（第4频道，Y轴）。创建栅极排除未染色或饱和荧光事件。打开两个直方图显示了原始最大像素为HLA-ABC（通道2）和鬼笔（第四频道）染色。 </li><li>在分析/面具菜单，科瑞EA新的面具，被称为从HLA-ABC染色细胞（通道2），侵蚀了2个像素的形态面具侵蚀（M02,2）。然后，在分析/功能菜单，创建包含圆参数的新功能，计算上的侵蚀（M02,2）。 注:此参数测量细胞形状的偏差的圆。因此，浑圆的T细胞将具有高圆形度的值，而细长偏振光细胞将显示出低圆度指数。 </li><li>接着，创建另一直方图，将来自先前门控细胞报告圆特征的值。使用这个直方图根据该圆度为每个单独的单元格的值直接绘制的个体，在焦细胞群的分布。 </li><li>看着直方图用于非刺激的细胞的形状，画出一个门，用于极化的细胞，从最低值圆到圆的限制值，其中大部分的未刺激细胞的绘制。看看统计数据显示极化细胞的门控人口的比例（％门控）。 注意:我们设置这个门为低于13圆的索引值。 </li><li>为了看在细胞中的肌动蛋白染色的偏振，画出的直方图为明亮详细相似度R3值，比较所述HLA-ABC染色（通道2）和鬼笔环肽染色（通道4）。 注:做大这一功能的价值，更多的叠加两个染色的。 </li><li>使用与以前相同的选通策略，在非刺激的细胞为分离的染色，显示细胞极化肌动蛋白染色的百分比绘制栅极。 </li><li>当完成时，细胞中的MHC I类和鬼笔环肽染色，偏光细胞连同所有细胞数±标准差的圆度和相似度指标的平均值的百分比的分布呈现偏析的百分比示于corresponding统计面板。 </li></ol>

<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>

作者宣称，他们有没有竞争的财务权益。

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突...

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><tbody><tr><td>RPMI</td><td>Gibco</td><td>61870-010</td><td></td></tr><tr><td>Human serum AB</td><td>PAA</td><td>C11-021</td><td>Pre-heat to inactivate the complement.</td></tr><tr><td>Fetal calf serum</td><td>PAN Biotech</td><td>P30-3300</td><td>Pre-heat to inactivate the complement.</td></tr><tr><td>Human T cell nucleofactor kit</td><td>Lonza</td><td>VCA-1002</td><td></td></tr><tr><td>Murine IL7</td><td>Peprotech</td><td>217-17</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>14025-050</td><td>Warm in 37 &amp;deg;C water bath before use.</td></tr><tr><td>Hepes</td><td>Gibco</td><td>15630-056</td><td></td></tr><tr><td>Murine CCL19</td><td>Peprotech</td><td>250-27B</td><td>Aliquots are thawed on ice before adding the chemokine to the cells.</td></tr><tr><td>Human CCL19</td><td>Peprotech</td><td>300-29B</td><td>Aliquots are thawed on ice before adding the chemokine to the cells.</td></tr><tr><td>Human CXCL12</td><td>Peprotech</td><td>300-28A</td><td>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><td>PFA</td><td>Electron Microscopy Sciences</td><td>157-8-100</td><td>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><td>BSA</td><td>Sigma</td><td>A3059</td><td></td></tr><tr><td>Saponin</td><td>Fluka</td><td>84510</td><td></td></tr><tr><td>Alexa Fluor 594 phalloidin</td><td>Invitrogen</td><td>A12381</td><td></td></tr><tr><td>FITC-anti-HLA-ABC antibody</td><td>Beckman Coulter</td><td>IM1838</td><td>clone B9.12.1</td></tr><tr><td>Anti-P-ERM antibody</td><td>Cell Signaling Technology</td><td>3149P</td><td></td></tr><tr><td>ImageStream&lt;sup&gt;x&lt;/sup&gt; Mark II</td><td>Amnis</td><td></td><td></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 ...

rapid-robust-analysis-cellular-molecular-polarization-induced

Universidad de Cartagena UDEC

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Immunology and Infection

Unit 1

Cell Polarization and Migration

SCIENTISTS IN ACTION

8.0K 次观看.  Institut Cochin. 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.

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

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

使用骨髓源性巨噬细胞巨噬细胞极化的调查

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

科研

免疫与感染

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

平面梯度扩散系统的调查趋化三维胶原基质

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

生物工程

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

细胞迁移研究在垂直化学和氧梯度聚二甲基硅氧烷的聚碳酸酯微流体装置

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

趋化梯度多细胞响应的三维分析

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

成像摹蛋白偶联受体（GPCR）介导的信号，控制趋化粘菌</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

生物学

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

ECIS的/出租车细胞趋化测量

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

外迁移系统中淋巴细胞迁移的评估

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a chemical. Chemokinesis and chemotaxis are fundamental for the mobilization and deployment of immune cells. For example, chemokines (chemotactic cytokines) can rapidly recruit circulating neutrophils and monocytes to extravascular sites of inflammation. Chemoattractant receptors belong to the large family of G protein-coupled receptors. How chemoattractant (i.e., ligand) gradients direct cell migration via G protein-coupled receptor signaling is not yet fully understood. In the field of immunology, neutrophils are popular model cells for studying chemotaxis in vitro. Here we describe a real-time two-dimensional (2D) chemotaxis assay tailored for mouse resident macrophages, which have traditionally been more difficult to study. Macrophages move at a slow pace of ~1 &#181;m/min on a 2D surface and are less well suited for point-source migration assays (e.g., migration towards the tip of a micropipette filled with chemoattractant) than neutrophils or Dictyostelium discoideum, which move an order of magnitude faster. Widely used Transwell assays are useful for studying the chemotactic activity of different substances, but do not provide information on cell morphology, velocity, or chemotactic navigation. Here we describe a time-lapse microscopy-based macrophage chemotaxis assay that allows quantification of cell velocity and chemotactic efficiency and provides a platform to delineate the transducers, signal pathways, and effectors of chemotaxis.

Here we describe methods using time-lapse, phase-contrast microscopy to image mouse resident peritoneal macrophages in a chemotactic complement C5a gradient. The protocols can be extended to other immune cells.

小鼠巨噬体化学术的延时成像

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

成像G蛋白偶联受体介导的趋化和信令活动在中性粒细胞样细胞HL60

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

对急性机械刺激的网柄反应的评估

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...