我们要感谢英于APC的最小/ +小鼠基因分型。这项研究是由来自INSERM基金和美国国立卫生研究院资助（CA057621和AI053194）的支持。

注：所有的动物实验均按照批准的机构动物照顾与使用委员会（IACUC），加州大学旧金山分校的程序进行。所有成像实验是非生存过程和动物立即安乐死图像采集结束后。 1代小鼠注：APC 最小/ +小鼠，携带突变的APC基因上，自发形成50-100腺瘤在小肠。 <ol><li>横装甲运兵车最小/ +小鼠与ACTB-ECFP线，其表达ECFP下，肌动蛋白启动子，并进一步与C-FMS-EGFP线，其表达EGFP下的c-fms的启动子来检测骨髓细胞。杂合子动物ACTB-ECFP和c-FMS-EGFP和用于检测荧光。 </li><li>用小鼠在3-4个月的婴儿图像清楚地发现腺瘤。 </li></ol> 2，准备的M材料板手术前注：在显微镜建立的细节描 ​​述6,7是以前。 <ol><li>显微镜<ol><li>打开加热毯。打开为488 nm激发和固态405纳米，561纳米和640纳米激光器的氩激光。打开显微镜，纺丝盘控制单元，所述声光可调滤波器，激光控制单元，相机控制器。 </li><li>打开显微镜快门，果然LED的红色。打开电脑和摄像头软件。 </li></ol></li><li>成像平台<ol><li>确保舞台的插入是干净的。否则，请用肥皂和水，然后擦干。 </li><li>举两个盖玻片，用实验室磁带，以确保他们的插件。将插入在舞台上，并用酒精擦拭清洁。 </li><li>使用实验室胶带，附加加热毯右侧的阶段。 </li><li>验证的橡胶隔膜的完整性上的鼻锥和如果necessar改变它年。位置和使用纱布和实验室磁带修复鼻锥的异氟醚麻醉分娩的动物。 </li></ol></li><li>异氟醚麻醉系统<ol><li>笔芯的异氟醚储罐。 </li><li>打开真空并调整到1.2升/分。 </li><li>加蒸馏水至雾化器和雾化器连接到异氟烷系统。 </li><li>打开氧气分析仪以及将其连接到异氟烷系统。打开氧气罐，并确保氧分析器表示100％。调节O 2流量0.2升/分钟。 </li><li>打开氮气罐和调节流量为0.8升/分。确保氧分析仪显示21％。 </li></ol></li><li>外科工具和平台的准备<ol><li>干净的2对解剖镊子和剪刀用肥皂和水。 </li><li>打开热珠灭菌并让它达到250℃。消毒的外科手术工具，持续30秒。让他们冷静下来。 </li><li>将实验室透雨手术板凳上。 </li><li>使用保丽龙盖的手术平台。盖上一块实验室透雨的。准备第二块实验室透雨的剃须后鼠标来改变。 </li><li>放置在鼻锥与麻醉有关的覆盖泡沫塑料盖的线，并将其与一些胶带附着到泡沫塑料盖（未在实验室中裂化反应室），并使用两针上的鼻锥的两侧，以将其固定到位。 </li><li>组装优碘，酒精擦拭，消毒纱布，强力胶，显微镜​​载玻片，电动剃须刀，4个实验室胶带。 </li></ol></li></ol> 3，制备注射剂<ol><li>引擎盖下，制备胰岛素注射器（28国祥½in）的7微升股票AF647标记的莱曼6G（Gr1的）抗体（1毫克/毫升）的入100μl的2000 kDa的罗丹明 - 葡聚糖（4毫克/毫升）在眼窝注射。眼窝注射，建议在装甲运兵车最小/ +小鼠由于贫血，在这些动物的发展，使尾静脉difficuLT检测通过皮肤。 </li><li>准备第二次胰岛素注射1毫克/公斤阿托品稀释成150μL的PBS皮下（SC）注射前成像挫伤肠道蠕动。 </li></ol> 4，准备在肠道的成像<ol><li>验证麻醉线到感应室是开放的，并且在线路的手术台和显微镜都关闭。 </li><li>动物转移到麻醉室。关闭麻醉室并打开异氟醚到4％（含21％O 2）。 </li><li>当鼠标深呼吸，并慢慢地（经过5-6分钟），将其传送到手术台上的侧翼和注入的Gr1抗体溶液和/或葡聚糖溶液眼眶。 </li><li>打开连接到外科平台的麻醉线。收线到麻醉室，把鼠标放在它与它的鼻子早在麻醉锥。 </li><li>减少异氟醚浓度从4％至2.5％。 </li><li>验证鼠标很好捏它的脚垫和测试角膜反射麻醉。 </li><li>添加眼科润滑膏对眼睛，以防止干燥时在麻醉下。 </li><li>加入实验室胶带固定鼠标的手术分期的四肢。 </li><li>通过使用电动剃刀除去从腹侧表面上的毛发。也从左侧后肢去除毛发以定位采集之前的血氧计。从手术分期取出实验室透雨，换上干净的，避免头发受到污染。 </li><li>消毒腹面用酒精湿巾和优碘。 </li><li>注射阿托品溶液（来自3.2）SC在小鼠中的一个侧面。 </li><li>请通过皮肤1厘米的腹正中切口及腹膜用剪刀和一个钳子。小心拉出3-4厘米小肠，并确定一个或多个肿瘤图像。 </li><li>乘坐玻璃MICRoscope需要滑动和位置就可以了肠道有肿瘤上方的循环。 </li><li>添加一些强力胶沿肠道几个地点将其附加到幻灯片。等待一分钟的胶水干。使用标记来画一条线在幻灯片上指向肿瘤，以促进其本土化的共焦。 </li></ol> 5，将鼠标在舞台上，并准备进行图像采集<ol><li>从四肢取出实验室胶带。 </li><li>打开麻醉线到显微镜载物台，并关闭所述一个到外科手术平台。 </li><li>仔细传输鼠标显微镜载物台，其腹面朝下，其在鼻锥鼻。安全与某些实验室带麻醉行。 </li><li>重新定位，以便在将盖子滑窗中的一个的中心的肠道的鼠标和/或滑动。轻轻大盘回落与实验室磁带的幻灯片。 </li><li>附测定仪的探针的鼠标的左肢到按照它的心脏和呼吸频率和血氧饱和度。 </li><li>涵盖了鼠标与加热毯，以避免体温过低。 </li><li>从2.5％降低异氟醚浓度为1-1.5％用于成像。 </li></ol> 6，图像采集利用μManager软件<ol><li>增加CSUX速度为2500 RPM，以提高在配置设置的图像质量。 </li><li>使用目标下拉菜单选择10X 0.5 NA或20X 0.75 NA Fluar透镜物镜。 </li><li>选择颜色下拉菜单中选择激光405纳米。 </li><li>点击Live和调整与显微镜的焦点。 </li><li>点击自动自动调节基于所述图像中的极端像素值的显示图像的最大和最小像素强度。显示图像的像素亮度直方图呈现在图表中的主窗口的下部。 </li><li>通过调整派珀控制面板窗口中的相机曝光改变时钟的数量（一个时钟是33.333毫秒）。 </li><li>如果该信号是太亮具有最低曝光，使用显微镜控制单元降低激光强度。 </li><li>检查信号强度为488纳米，561纳米和640纳米的激光线。调整激光强度与激光自身的控制单元（488纳米和561纳米）或显微镜控制单元。 </li><li>在多D采集窗口，检查时间点框，并输入时间点数目和所需的时间间隔。 </li><li>检查Z-堆栈中，选择“相对Z”和定义的Z开始和相对Z-端的当前位置。 注：为10X，20X或目标，除了分别开始3 Z平面，8微米或4微米。 </li><li>检查多个位置（XY）中成像的几个地点，然后单击编辑位置列表中，选中4至6个不同的位置。 </li><li>检查通道中，并添加来自新的渠道和选择激光从下拉菜单和典型线路E在曝光对于每个信道（在33.333毫秒的倍数）。 </li><li>检查影像保存在“多D采集”窗口。 </li><li>选择一个文件夹，所有采集的图像会被自动保存在这个文件夹中特定的名称前缀。 注：每个连续的收购将被保存在一个单独的子文件夹作为单独的TIFF文件中的每个颜色，每个时间点每个Z片。 </li><li>通过点击多D采集窗口保存设置保存所有设置。 </li><li>单击收购多维采集窗口，开始采集。 </li><li>如果测定仪的探针不使用或停止工作以及（当脉冲是不稳定或难以检测），通过夹持足垫和检查角膜反射检查麻醉的效率的摄像过程中，每隔15分钟。调整麻醉，如果鼠标响应这些刺激。 </li><li>采集后，通过提高异氟烷concent安乐死的小鼠配给到5％。如果没有呼吸动作检测，从舞台移开鼠标，并进行颈椎脱位。 </li></ol>图像利用了Imaris软件分析7。 <ol><li>转换到.ims文件（ 补充图1A） <ol><li>打开摄像头软件，点击文件，然后批量转换。 </li><li>点击添加文件，选择第一个位置的第一个文件。重复每个位置上。 </li><li>对于上传的每个文件，点击设置，验证设置“T”代表时间，“C”的通道和“z”为Z-堆栈中出现的顺序。 </li><li>保存在指定的文件夹。浏览并转化为创建一个新的文件。 </li><li>点击设置为所有和启动。 </li></ol></li><li>调整（ 补充图1B） <ol><li>进入具体的转换文件夹，打开的第一个文件。 </li><li>在显示调整窗口，调整背景通过在必要时修改的最小数目为每个信道的信号截止。 </li><li>如果需要的话，通过修改最大数目（下的最大数量，较高的信号的强度）调节的信号强度。 注：信号强度/对比度调整使细胞行为或隔间的亮点，才能有一个更好的可视化。它不改变结果本身。 </li><li>点击编辑和图像属性。 </li><li>改变的x，y和z的值（为一个10X物镜，x和y = 0.712，Z = 8微米;对于20X物镜：x和y = 0.36，Z = 4微米）。 </li><li>点击所有等距调整的时间点。添加的开始日期和开始采集时间。添加结束日期和结束收购（ 补充图1C）的时间。 </li><li>点击播放按钮观看影片。 </li><li>点击编辑和删除的时间点，删除图像模糊。 </li><li>加入两个单独收购峠疗法为一体的电影，去的第一部电影的最后一个时间点，然后点击编辑&gt;添加的时间点，添加第二个文件。 </li></ol></li><li>保存为电影（ 补充图1D） <ol><li>点击记录，选择.MOV延伸和0的压缩因子。 </li><li>单击Save（保存）。 </li></ol></li></ol>

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The authors have nothing to disclose.

在本文中，详细的协议描述中纺肠道肿瘤骨髓细胞动力学盘共聚焦成像几个小时的活的动物，从肠道的浆膜面成像。 避免炎症反应，有最佳的生理条件下，在肠道的摄像，必须在完整器官进行。然而，从肠的浆膜侧成像是具有挑战性的，由于光具有到达上皮之前要经过不同的组织层，例如平滑肌。着眼于上皮可以在某些方面是困难的，特别是在肌动蛋白-ECFP报告小鼠由于肌动...

大量证据现在表明，在肿瘤微环境，包括异质细胞群，包括成纤维细胞，内皮细胞，免疫和炎症细胞，细胞外基质，和可溶性因子，通过促进几乎所有起着起始和实体瘤的进展的关键作用癌症1标志。的确，在肿瘤进展，也有发展，以产生微环境有利于恶性2转化的肿瘤细胞和基质细胞之间恒定的动态相互作用。间的免疫细胞浸润的肿瘤微环境，骨髓细胞是最丰富3。肿瘤相关巨噬细胞中的组成（TAM），髓源抑制细胞（肌源性干细胞），树突状细胞（DC）和嗜中性粒细胞（中性粒细胞），骨髓细胞从骨髓中招募和渐进渗透的肿瘤，释放出细胞因子，生长因子和蛋白酶的可以促进肿瘤的生长和扩散4。肿瘤细胞和骨髓细胞之间的串扰是复杂的，而是动态的。因而它们之间的相互作用的性质的认识是用于确定为什么这些细胞促进癌症的进展，而不是参与抗肿瘤免疫应答的关键，并且可以有助于发现新的目标，以控制它。 直接观察活体显微镜提供了活体小鼠5的组织中细胞动力学信息。四色，多区域，微透镜式旋转盘共聚焦系统被设计成乳腺肿瘤6中学习基质细胞。这种方法使长期连续成像，并包括若干优点，诸如（a）快速图像获取，以减少运动伪影，（二）长期麻醉，（三）四种颜色的采集遵循不同的细胞类型，（四）的荧光标记不同肿瘤部件，和（e）观察的不同肿瘤微环境机智欣相同的鼠标，避免鼠标鼠标的变化7-9。通过该技术，不同的细胞行为已有报道在乳腺肿瘤病毒（MMTV）启动子驱动的多瘤病毒中间Ť癌基因（PyMT）模型，该模型显示肿瘤发生的渐进阶段。调节性T-淋巴细胞（Treg细胞，由Foxp3的EGFP转基因的可视化）优先在接近迁移到血管而树突（CD11c的-DTR-EGFP），癌相关成纤维细胞（FSP1 + / +-EGFP）和髓样细胞（C-fms的-EGFP）显示出更高的运动在肿瘤周边比肿瘤块内。急性全身缺氧的条件下，细胞迁移是不同的：停止调节性T细胞相反，骨髓细胞是继续往前6迁移。此外，在相同的小鼠模型中，已经显示与肿瘤阶段的多柔比星的敏感性的变化，药物的分布与药物的反应，以及多柔比星处理导致CCR2依赖招募骨髓细胞的肿瘤。因此，实时成像，也可用于深入了解药物反应的原位和耐药10,11的生物。 性腺瘤性息肉病（APC）基因突变通常发生在人类结直肠腺瘤和癌12的APC基因导致家族性腺瘤性息肉病（FAP），它赋予了极高的风险结肠癌13的一个副本变异。在小鼠品系装甲运兵车 最小 / +进行截断突变的APC基因的密码子850和自发发展多种肠道腺瘤各地小肠14-16。肠的长期活体成像是因为该过程的侵袭的具有挑战性的，因为在打开腹腔是必要的访问权限的肠。短期实时成像STudies先前已公布在健康肠17,18，但长期直接观察肠道肿瘤尚未见报道。一种外科手术已被设计和精制通过肠道的浆膜表面显现肿瘤，使用先前用于图像的乳腺肿瘤6,10的活体旋转盘显微镜系统。在本文中，一个协议描述了允许一个由通过 APC调节最小 / +小鼠跟随在小肠中的肿瘤内的髓样细胞的行为。

<table border="0" cellpadding="0" cellspacing="0" width="691">
	<tbody>
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			<td height="42" style="height:42px;width:216px;"><a name="RANGE!A1:D30">Name of Material/ Equipment</a></td>
			<td style="width:107px;">Company</td>
			<td style="width:141px;">Catalog Number</td>
			<td style="width:227px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ApcMin/+ mice</td>
			<td style="width:107px;">Jackson Laboratory</td>
			<td>2020</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ACTB-ECFP mice</td>
			<td style="width:107px;">Jackson Laboratory</td>
			<td style="width:141px;">3773</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">cfms-EGFP mice</td>
			<td style="width:107px;">Jackson Laboratory</td>
			<td align="right">18549</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2,000 kDa Dextran, rhodamine-conjugated</td>
			<td style="width:107px;">Invitrogen</td>
			<td style="width:141px;">D7139</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Isoflurane</td>
			<td style="width:107px;">Butler Animal Health Supply</td>
			<td style="width:141px;">29450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nitrogen</td>
			<td style="width:107px;">UCSF</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oxygen</td>
			<td style="width:107px;">UCSF</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1X PBS</td>
			<td style="width:107px;">UCSF cell culture facility</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Saline Buffer</td>
			<td style="width:107px;">UCSF cell culture facility</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-mouse Ly-6G (GR1) antibody AF647</td>
			<td style="width:107px;">UCSF Monoclonal antibody core</td>
			<td style="width:141px;"></td>
			<td>Stock 1mg/ml. Use at 7ug/mouse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Atropine</td>
			<td style="width:107px;">LARC UCSF</td>
			<td style="width:141px;"></td>
			<td>Use at 1mg/Kg mouse</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alcohol wipes</td>
			<td style="width:107px;">Becton Dickinson</td>
			<td style="width:141px;">326895</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">28G1/2 insulin syringe</td>
			<td style="width:107px;">Becton Dickinson</td>
			<td style="width:141px;">329465</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Remium cover glass</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:141px;">12-548-5M</td>
			<td>24x50-1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Superfrost plus microscope slides</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:141px;">12-550-15</td>
			<td>25x75x1mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Krazy glue</td>
			<td style="width:107px;">Office Max</td>
			<td align="right">7111555</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Betadine</td>
			<td style="width:107px;">LARC UCSF</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Heat blanket</td>
			<td style="width:107px;">Gaymar Industries</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hot bead sterilizer</td>
			<td style="width:107px;">Fine Science Tools</td>
			<td style="width:141px;">18000-45</td>
			<td>Turn ON 30min before use</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cotton tipped apllicators 6-inch</td>
			<td style="width:107px;">Electron Microscopy Sciences</td>
			<td style="width:141px;">72310-10</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anesthesia system</td>
			<td style="width:107px;">Summit Anesthesia Support</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted microscope</td>
			<td style="width:107px;">Carl Zeiss Inc</td>
			<td style="width:141px;">Zeiss Axiovert 200M</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">stage insert</td>
			<td style="width:107px;">Applied Sientific Instrumentation</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mouse Ox oximeter, software and sensors</td>
			<td style="width:107px;">Starr Life Sciences</td>
			<td style="width:141px;">MouseOx</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nebulizer</td>
			<td style="width:107px;">Summit Anesthesia Support</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Imaris</td>
			<td style="width:107px;">Bitplane</td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mManager</td>
			<td style="width:107px;">Vale lab, UCSF</td>
			<td style="width:141px;"></td>
			<td>Open-source software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ICCD camera</td>
			<td style="width:107px;">Stanford Photonics</td>
			<td style="width:141px;">XR-Mega-10EX S-30</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spinning disk confocal sacan-head</td>
			<td style="width:107px;">Yokogawa Corporation</td>
			<td style="width:141px;">CSU-10b</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time imaging of myeloid cells dynamics in apcmin/+ intestinal tumors by spinning disk confocal microscopy

髓系细胞是在肿瘤内最丰富的免疫细胞和已被证明能促进肿瘤进展。现代活体成像技术使观察脏器内部活细胞行为的，但可以在一些类型的癌症，由于器官和肿瘤的辅助具有挑战性，例如肠。直接观察肠道肿瘤以前尚未报道。这里描述的手术过程可直接观察活体小鼠肠道内的肿瘤骨髓细胞动力学的利用转基因荧光报告小鼠和注射示踪剂或抗体。为了这个目的，一台四色，多区域，微透镜式旋转盘共聚焦显微镜，允许长期连续成像的快速图像捕获已被使用。 装甲运兵车 最小 / +小鼠的发展在小肠多发性腺瘤是正交与C-FMS-EGFP小鼠可视化骨髓细胞和ACTB-ECFP老鼠以可视化的隐窝肠上皮细胞。用于标记不同肿瘤成分，如血管和嗜中性粒细胞，并通过浆膜表面定位的肿瘤进行成像的方法的程序也被描述。从几个小时影像编定时短片允许髓细胞行为的原位肠道微环境的分析。

通过使用旋转盘共聚焦显微镜，非肿瘤和肿瘤组织在小肠装甲运兵车最小/ +的; ACTB-ECFP;的c-FMS-ECFP小鼠可以从浆膜表面可视化。成像后，照相机软件用于分析和调整的采集（ 附图1）。静脉注射（IV）注射荧光2000 kDa的葡聚糖-罗丹明B和莱曼6G 647结合的抗体后，血管和中性粒细胞可分别检测到（ 图1）。淋巴集结是淋巴组织的聚集和全骨髓细胞可?...

Watch this Scientific Journal Video about Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy at JoVE.com

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

real-time-imaging-myeloid-cells-dynamics-apcmin-intestinal-tumors

Research

JoVE Journal

Immunology and Infection

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

9.6K 次观看.  INSERM U661, Functional Genomic Institute. By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model. 

视频: Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

活体显微镜的脾：寄生虫的流动性和血流量的定量分析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

科研

免疫与感染

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 ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

分析<em&gt;小肠结肠炎耶尔森菌</em&gt;效应易位到宿主细胞中使用β-内酰胺酶效应融合体

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.