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>

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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触12的上下文分析分子再分配事件。此外，一组类似的向上已经用于SDF1刺激T细胞但已被提供13没有分析共定位的两个标记之间的程度。 虽然因而这种技术可以适用于许多类型的细胞，粘附细胞，不能直接进行分析和细胞悬浮液之前需要采集固定。除了机器的这些固有的限制，这是关键的刺激它们时，以保持细胞在37℃。温度的确键实现合适的T细胞的极化自细胞骨架的变化，如肌动蛋白聚合，驱动形态的变化，都非常依赖于温度。我们的实验用latrunculin A处理的细胞确认正确的肌动蛋白重塑诱导趋化因子的刺激确实是至关重要的一个单元，以实现其完全极化状态。除了提供这里用此药的数据，我们还测试了成像流式细胞仪技术与转染了该抑制PAR6 /蛋白激酶Cζ信号通路的突变体蛋白的人原代T细胞。始终与我们先前发表的结果如图7所示 ，它在这里显示，该极性复合氟avors T细胞极化。 在趋化因子的刺激，T细胞失去一轮的形态采用偏光形状。我们已经找到了圆度参数是最准确量化这些形态学改变。然而，描述的形状的改变其它参数提出的分析软件，如长宽比或Elongatedness。因此，人们可以很容易地测试特定信号传导途径的或在此过程中的贡献的特定蛋白质。 在这项研究中，我们一直观察到，细胞表现出形态偏振的比例比1分子极化更高。这表明细胞采用形态学的变化不存在的标记大分子再分配的一小部分研究。这可能源于一个事实，即有些细胞失去圆形状，使其具有较低的圆度指数虽然他们并没有明确显示出导入区克边缘和尾肢。这些部分偏振的T细胞可以仍然落入在形态上极化T细胞的栅极没有呈现的典型标志物分子明显再分配。此外，在趋化因子信号传导的阈值的差异，可能是更高的标记再分配比形状的改变也可以解释这种差异。 它也在这里显示了HLA-ABC标志未relocalized在极化T细胞和有在该染色后CCL19刺激强度没有变化。相反，确认在此提供的F-肌动蛋白染色的增加后CCL19的治疗（ 图1和2）如已经公知的，由于趋化因子刺激激活肌动蛋白聚合5。另外，它表明，肌动蛋白微丝得到在极化细胞的前relocalized。因此，明亮的细节相似度指数是衡量两者之间的马共定位的程度rkers趋向于CCL19刺激减少。作为比较，这样的分析是在这里提供的F-肌动蛋白的共定位与磷酸化的ERM蛋白的程度获得排除在电池正面。趋化因子刺激的T细胞表现出丝状肌动蛋白的百分比- MHC I类偏析比为1 F-肌动蛋白低得多-磷酸ERM（14％比分别为48％，），因为在前面的F-肌动蛋白和relocalizes磷酸化的ERM蛋白在极化T细胞的后部，而MHC I类分子的再分配不受趋化因子刺激。这间明亮的细节相似度指数因此可以系统地用来比较两个指标的极化T细胞的分布。因此，这种技术是非常合适的量化共定位或共排斥两种不同的标记的程度上有大量的细胞或潜在的，存在于广泛的细胞群罕见的事件。

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。 首先，将圆度指数测量在未处理的（ 图1）或CCL19刺激（ 图2）的T淋巴细胞。该静息细胞有具有高圆形度指数值的一个主要的单峰的事实表明，大部分细胞是接近圆。然而，很明显，CCL19刺激诱发的外观Ò细胞FA具有低圆指数第二亚群。该亚群包括已采用极化形态的细胞。此外，代表HLA-ABC和F-肌动蛋白染色的明亮详细相似度索引时，可以观察到某些细胞表现出在此指数时CCL19的治疗价值的下降，这表明这两个标记显示共定位的程度较低。有趣的是，从点图先前提出，可以在任何特定的点的位置的光标来获得由一个明图像和每个进行染色的图像的细胞的照片。 图3从而呈现CCL19的例子刺激的T淋巴细胞落在非偏振光（ 图3A）或极化（ 图3B）细胞的栅极。人们可以清楚地可视化亚两者之间的差异在形态。从下面的圆度直方图的统计信息的表，从图1 </strong&gt;和2中 ，人们可以绘制细胞的落在偏振光栅极在没有或CCL19的存在（ 图3C）的百分比。正如预期的那样，很明显，趋化因子刺激的增加极化T细胞的百分比（从11.2％到50.6％）。人们也可以绘制在两个条件（ 图3D）中获得的全细胞群体的平均圆度指数。 CCL19刺激诱发这一指数下降。然而，由于只有一半的细胞被极化，在后CCL19刺激观察到的平均圆形度指数的变化是小的。 类似地， 图4表示CCL19刺激的细胞，在其中两个HLA-ABC和F-肌动蛋白染色做（ 图4A），或者不共定位（ 图4B）的示例。从下面的详细光亮相似直方图的统计信息的表在图1和2所示，可以得到比重细胞表现出低于1.5的指数值，代表表现出非常低的HLA-ABC和F-肌动蛋白之间的共定位的程度的细胞。 图4C显示了细胞的落入在后CCL19刺激这个门增加分数（从3.4％到13.9％）。收集指数为每个单独的小区的值，还可以绘出在两个条件下（ 图4D）中获得的全细胞群体的平均明亮详细相似度索引。 CCL19引出在指数下降，但是，由于同样的原因如前面，变化小。通过查看对应细胞的图像，看来这一下降在HLA-ABC - F-肌动蛋白的共定位，主要是由于F-肌动蛋白的细胞中的一极的聚类。相反地​​，似乎HLA-ABC MHC I类分子的细胞内的分布不被严重受CCL19刺激。 这就是为什么我们决定明年进一步测试的原因通过分析两个标记（F-肌动蛋白和磷酸化ERM）众所周知的方法来隔离在反对趋化因子刺激T细胞的两极。人类T细胞与CXCL12刺激强烈极化，如量化， 如图5A与同门控策略比以前（从5.95％至79.10％，偏振光细胞刺激后的用50ng / mL的CXCL12 8分钟）。在这些条件下，有如图5B和5D所示 ，这意味着F-肌动蛋白和磷酸化的ERM共定位CXCL12刺激后较少在平均亮度细节相似度索引一个强烈减少。正如预期的，细胞表现出在这里所选择的标记物的偏析的百分数较高（48％， 图5C）相比先前研究的那些（14％， 图4C）。因此，在各条件下的平均相似性指数的下降也更明显（ 图5D）。因此，这里提出的成像流式细胞仪技术是苏itable量化共定位在不同的设置2染色程度。 最后，我们决定以说明在什么程度上一些细胞骨架元件或信号通路已知影响时的趋化因子刺激的变化的T细胞形态学的扰动，可以通过成像来测定流式细胞术。控制T细胞首先与一些预处理的肌动蛋白破坏药物latrunculin A（ 图6A）相比较。细胞用CXCL12，由成像流式细胞染色用荧光鬼笔环肽进行分析。直方图表示F-肌动蛋白染色的原最大像素强度示于两种细胞群（ 图6A，上图）。正如预期的那样，从latrunculin朝向微丝A的切割活性，所述鬼笔环肽染色的强度低在这些细胞中（右图）相比，控制与车辆的DMSO（左图）处理的T淋巴细胞。此外，latrunculin A-TReated T细胞表现表明，他们仍然比圆对照细胞（ 图6A，底部）一个更大的圆度指数。这可以通过栅极上的直方图设定为测量细胞表现出形态学改变的百分比来定量。虽然对照细胞的32％得到偏振光在这种条件下，只有10％的latrunculin甲处理过的T淋巴细胞表现出任何形状的变化（ 图6B）。因此，它在这里显示了流式细胞技术的摄像流量适合测量形态学修饰的差异，当一个研究了在这种现象的一些细胞骨架元素的潜在影响。 其次，获得了大量的细胞用这种方法也将打开的可能性，以分析细胞存在作为少数的快速小的子集。在图7中 ，一个例子给出，其中我们的分析是特别的限制，以T细胞的共转染的行为ED与GFP与质粒极性复杂PAR6 /PKCζ的显性负突变体的编码。对应于非转染的细胞的直方图，使我们能够栅极专门对相应的GFP + T细胞（ 图7A，左）。的GFP测定作为量化的信道2的荧光强度的分布直方图的强度确实显示，全细胞群体的一小部分已被转染的（ 图7A）。这些转染的细胞的形态偏振度，然后通过测量的GFP +细胞（ 图7B）的唯一的圆度指数定量。对于T细胞表现出圆度值低的附加 ​​栅极允许估计极化细胞在每种条件下（ 图7C）的百分比。的结果表明，该PAR6 /蛋白激酶Cζ信号通路的功能的破坏抑制T细胞polarizati由双方CCL19和CXCL12趋化因子诱导，符合我们先前的调查结果7。 <img alt="图1" src="/files/ftp_upload/52140/52140fig1.jpg" width="650" /> 图上的原代人T细胞1.代表性的非刺激的染色结果。左上面板显示梯度的RMS的直方图再分配与在明视场图像中的M01掩模计算（通道1）。右上面板显示M01掩模侵蚀的2个像素的基础上，明的图像的焦点。在细胞从先前情节选通的区域（通道1）。中间面板显示与HLA-ABC和鬼笔环肽染色两者（分别通道2和4）无论是作为一个散点图（左图）或作为直方图（中间和右面板），用于个人的生最大像素值，在 - 集中在上一步中选择单元格。从个别，对焦细胞正确的染色这一人群，<html>被计算出的圆形度特征在M02掩模侵蚀的2个像素的HLA-ABC染色（左下图）和在明亮的详细相似度R3特征（右下图）示出了HLA-ABC和鬼笔环肽染色之间的相似度的。对于这两个地块的最新统计表明人口的重新分配（％门，意味着该功能，SD）的指示在下面的表格。 <a href="https://www.jove.com/files/ftp_upload/52140/52140fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52140/52140fig2.jpg" width="650" /> 图2:对人原代T细胞用200纳克/毫升的CCL19 8分钟刺激代表染色结果的面板布局和门控策略是等同于图1。JPG"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/52140/52140fig3highres.jpg" /> 图3:由CCL19刺激的T淋巴细胞的形态学状态量化。 （A，B）细胞从图2的外部，或在"极化"门的实例，分别为（C）的柱状图示出了在不存在CCL19的所得偏振性T细胞的百分比。（ 图1， - ）或用200纳克/毫升CCL19（ 图2，+）。（D）的直方图表示在各个圆形指数±标准的平均值，在焦细胞群在不存在- CCL19的或存在（+）（）。 *** P &lt;0.001。 <a href="https://www.jove.com/files/ftp_upload/52140/52140fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a><img alt="图4" src="/files/ftp_upload/52140/52140fig4highres.jpg" /> 图4.定量的HLA-ABC和毒伞素染色之间的合作，本地化的程度。 （A，B）细胞从图2的外部或在从明亮详细相似度直方图，分别为"不共定位"门的例子。（C）的直方图表示T细胞表现出两个标记在非之间没有共定位的百分比刺激（ - ）。或CCL19刺激（+）的条件（D）的直方图表示在个体相似性指数±标准的平均值，在焦细胞群在不存在- CCL19的或存在（+）（）。 *** P &lt;0.001。 <a href="https://www.jove.com/files/ftp_upload/52140/52140fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 对together.within页="总是"&gt; <img alt="图5" src="/files/ftp_upload/52140/52140fig5highres.jpg" /> F-肌动蛋白的图5.分析-磷酸ERM排除对人原代T细胞的CXCL12刺激。极化T细胞在CXCL12（10或50纳克/毫升）的存在或不存在（A）中所占的百分比。（B）的成像流式细胞直方图示出了个人，在焦细胞群根据布赖特详细相似度索引的重新分区细胞没有表现出共定位的比例，用于比较磷酸化的ERM和鬼笔环肽染色之间的共定位的程度，在不存在CXCL12的或用10ng / ml或50ng / ml的CXCL12。（C）的定量烫发和T细胞的毒伞素染色刺激或不CXCL12。相似度指数±SE在每个条件的平均值（D）量化。 *** P &lt;0.001。上传/ 52140 / 52140fig5large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图6" src="/files/ftp_upload/52140/52140fig6highres.jpg" /> 肌动蛋白聚合对T细胞极化图6.影响。 （A）的 F-肌动蛋白与Latrunculin A（500纳米，30分钟）或DMSO处理，然后初级人T细胞的染色强度（上图）和圆度（下面板）刺激用100ng / ml的CXCL12 8分钟。（ B）直方图显示与DMSO或Latrunculin A之后刺激CXCL12的8分极化处理的T细胞的百分比。 <a href="https://www.jove.com/files/ftp_upload/52140/52140fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/52140/52140fig7.jpg" width="650" /> 图中的蛋白激酶Cζ激酶死突变体或PAR6块T细胞极化后CCL19或CXCL12治疗的N-末端的突变体的7的表达。 （A）的成像流式细胞直方图表示GFP表达强度，在非转染的初级T细胞或原代T细胞共转染GFP和空载体（对照）或蛋白激酶Cζ的激酶死突变体或N末端PAR6的部分（B）成像流式细胞仪代表GFP阳性细胞群体表达的不同构建体在基础水平的圆度指数直方图（NS:无刺激）。或CCL19或CXCL12刺激（100之后纳克/毫升8分钟）。（C）的形态极化T细胞表达不同的结构的基础水平或CCL19或CXCL12刺激后的百分比量化。 <a href="https://www.jove.com/files/ftp_upload/52140/52140fig7highres.jpg" target="_blank">请点击此处查看大版本</a>锡安这个数字。

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 Científica del Sur

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JoVE Journal

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Cell Biology

Immunology and Infection

Unit 1

Cell Polarization and Migration

SCIENTISTS IN ACTION

8.1K 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

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

低氧环境下人巨噬细胞极化的蛋白质组学分析

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

科研

生物化学

Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury

After brain stroke microglia/macrophages (M/M) undergo rapid activation with dramatic morphological and phenotypic changes that include expression of novel surface antigens and production of mediators that build up and maintain the inflammatory response. Emerging evidence indicates that M/M are highly plastic cells that can assume classic pro-inflammatory (M1) or alternative anti-inflammatory (M2) activation after acute brain injury. However a complete characterization of M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is still missing.	
	Immunofluorescence protocols specifically staining relevant markers of M/M activation can be performed in the ischemic brain. Here we present immunofluorescence-based protocols followed by three-dimensional confocal analysis as a powerful approach to investigate the pattern of localization and co-expression of M/M phenotype markers such as CD11b, CD68, Ym1, in mouse model of focal ischemia induced by permanent occlusion of the middle cerebral artery (pMCAO). Two-dimensional analysis of the stained area reveals that each marker is associated to a defined M/M morphology and has a given localization in the ischemic lesion. Patterns of M/M phenotype marker co-expression can be assessed by three-dimensional confocal imaging in the ischemic area. Images can be acquired over a defined volume (10 &mu;m z-axis and a 0.23 &mu;m step size, corresponding to a 180 x 135 x 10 &mu;m volume) with a sequential scanning mode to minimize bleed-through effects and avoid wavelength overlapping. Images are then processed to obtain three-dimensional renderings by means of Imaris software. Solid view of three dimensional renderings allows the definition of marker expression in clusters of cells. We show that M/M have the ability to differentiate towards a multitude of phenotypes, depending on the location in the lesion site and time after injury.

A way to gain new insights into the complexity of the brain inflammatory response is presented. We describe immunofluorescence-based protocols followed by three-dimensional confocal analysis to investigate the pattern of co-expression of microglia/macrophage phenotype markers in a mouse model of focal ischemia.

偏振的实验性脑损伤小胶质细胞/巨噬细胞标志物的三维共聚焦分析

After brain stroke microglia/macrophages (M/M) undergo  rapid activation with dramatic morphological and phenotypic changes that  include expression of ...

神经科学

An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing to the transparent embryonic body of zebrafish, it is convenient for us to study the effect of one chemokine on the behavior of a certain type of cells in vivo. Here we provided a workflow to investigate the function of a chemokine on macrophage migration in vivo. We constructed a tissue-specific overexpression plasmid to overexpress IL-34 and injected the plasmid into one-cell stage transgenic fish embryos whose macrophages were specifically labeled by a fluorescent protein. We then used whole mount fluorescent in situ hybridization and immunostaining to detect the pattern of the chemokine expression and the number or location of macrophages. The injected WT embryos were raised to generate a stable transgenic line. Finally, we used confocal live imaging to directly observe macrophage behavior in the stable transgenic fish to study the function of IL-34 on macrophages in vivo.

To test the effect of a chemokine on macrophage recruitment in vivo, the whole mount in situ hybridization was used to detect the ectopic expression of the chemokine, and immunostaining was used to label macrophages. Live imaging was used for real-time observation of macrophage migration.

用于测试 Vivo 中巨噬菌体招募的异位化学表达模型

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing ...

发育生物学

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

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

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 ...

免疫与感染

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

一种直接从一滴血液中快速中性粒细胞趋化分析的片上方法

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

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 ...

生物工程

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

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

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 ...