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 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。 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 San Sebastian

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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

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

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors1. This process leads to bacterial invasion of endothelial cells2. More recently, we observed an interaction of Opc with a 100kDa protein found in whole cell lysates of human cells3. We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as &alpha;-actinin. As no surface expressed &alpha;-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and &alpha;-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with &alpha;-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

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

Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human &alpha;-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular &alpha;-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).

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

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

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

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

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

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

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 ...

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

一个钙生物发光检测蚊虫的功能分析（埃及伊蚊）和刻度（镰形扇头microplus）G蛋白偶联受体

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

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

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

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

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

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

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

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

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

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

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

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 ...

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

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

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

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

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 ...

细胞与分子极化的快速和强大的分析趋化因子诱导信号

摘要

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此视频中的章节

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

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

滴流和旋转盘反应器使用金黄色葡萄球菌生物膜分析

一个钙生物发光检测蚊虫的功能分析（埃及伊蚊）和刻度（镰形扇头microplus）G蛋白偶联受体

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

一个实时，同步成像的TCR和其相关的信号转导蛋白的TIRF显微镜技术

上皮损伤制作分析

在小鼠B淋巴细胞染色体畸变所PNA-FISH快速分析

快速分子检测和流感病毒A和B的分化

分子互补亲和纯化 (BiCAP) 对多蛋白信号络合物的解剖

细胞与分子极化的快速和强大的分析趋化因子诱导信号

摘要

探索更多视频

此视频中的章节

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

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

滴流和旋转盘反应器使用金黄色葡萄球菌生物膜分析

一个钙生物发光检测蚊虫的功能分析（埃及伊蚊）和刻度（镰形扇头microplus）G蛋白偶联受体

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

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

上皮损伤制作分析

在小鼠B淋巴细胞染色体畸变所PNA-FISH快速分析

快速分子检测和流感病毒A和B的分化

分子互补亲和纯化 (BiCAP) 对多蛋白信号络合物的解剖

一个实时，同步成像的TCR和其相关的信号转导蛋白的TIRF显微镜技术