这项工作由美国国家癌症研究所（NCI）拨款U54CA143798和U54CA143907资助，分别在达纳 - 法伯癌症研究所和南加州大学建立物理科学 - 肿瘤学中心（PS-OC）。 SM Mumenthaler获得了PS-OC跨网络奖，支持了这项工作。 衷心感谢我们的慈善支持者，特别是Stephenson家族，Emmet，Toni和Tessa，他们捐赠了Operetta HCS平台。我们还要感谢J. Foo指导，应用分子医学中心团队成员:D. Agus临床指导和指导，K. Patsch与实验设计进行有意义的讨论，R. Rawat为图像分析协议提供帮助，J. Katz与Operetta的技术协助，以及P. Macklin和D. Ruderman进行了有益的讨论和反馈。

细胞培养注意:在与肺成纤维细胞（CCD-19Lu GFP）共同培养的情况下，研究NSCLC细胞（H3255）对EGFR靶向药物埃罗替尼的表型反应。对于相同的数据集，进行了两个细胞群体（H3255和CCD-19Lu GFP）的基于荧光和基于形态的分类，以证明它们的一致性。然而，只需要使用一种分类方法，并应根据应用选择。 <ol><li>准备500毫升补充有10％热灭活FBS和1％青霉素/链霉素的RPMI-1640。 注意:生长培养基和补充剂可以根据其他细胞类型的要求进行更换。 </li><li>培养细胞在10cm 3平板中作为纯种群在5％CO 2中于37℃，并以适当的比例每3-4天通过。 </li></ol> 2.细胞的制备<ol><li>要制备细胞悬液，取出c并用5mL 1X磷酸盐缓冲盐水（PBS）洗涤细胞。 </li><li>从PBS中吸出，加入1 mL加入到37°C的0.05％胰蛋白酶，并在37℃下孵育5分钟（或直到细胞不再粘附于平板）。 </li><li>为了中和胰蛋白酶，向H3255和CCD-19Lu GFP板中加入4mL RPMI，并将细胞溶液移至15mL锥形管中。 </li><li>在室温下以150×g离心细胞5分钟。 </li><li>将上清液吸出并将细胞沉淀重悬于10mL RPMI培养基的锥形管中以准备播种。 </li></ol>电池电镀<ol><li> 计数细胞以使种子标准化。 <ol><li>反转管将溶液中的细胞混合。将10μL每个细胞悬浮液移至微量离心管中，并加入10μL台盼蓝。 </li><li>将10μL此溶液移至细胞计数载玻片并插入自动细胞计数器。 注意:其他细胞计数方法（ 即。血细胞计数器）。 </li><li>重复细胞计数至少三次，或直到获得的计数一致。 </li></ol></li><li> 将种子细胞置于多孔板上 注意:种子的数量取决于将被成像的时间点数。或者，如果细胞用组蛋白-2B-GFP（或提供足够的核分割的其他稳定的慢病毒核染色体）转导，则可以随时间重新成像一个平板。 <ol><li>在100μLRPMI中种子总共1500个细胞/孔。准备三种细胞悬浮液来平板三种细胞群体:H3255，CCD-19Lu GFP和50％H3255 + 50％CCD-19Lu GFP。对于每个时间点，使用相同的板设置执行一式三份。 注意:应针对每个细胞系和板尺寸优化初始播种密度。 </li><li>使用多通道移液器在3×96孔板中的种子细胞。在37℃，5％CO 2下孵育细胞O / N。 </li></ol> &lt;/ LI&gt; </ol>药物剂量<ol><li>将DMSO加入厄洛替尼粉末中以制备10mM浓度的最终埃罗替尼储备溶液。 注意:药物可以根据需要进行取代。 </li><li>用细胞培养基稀释药物至最终浓度的2倍，最终浓度为10μM，以1:10的比例连续稀释4次，共5种药物浓度和无药物对照。 注意:如果最高药物剂量的DMSO浓度等于或超过0.1％v / v，则无药物对照应含有等量的DMSO，以确保观察到的观察效果不是由于DMSO毒性所致。 </li><li>将100μL药物溶液移至相应的孔中，以最终药物浓度1×稀释于培养基中。 </li></ol>图像采集注意:在第0,2和3天获得图像。根据正在研究的细胞类型和细胞过程，o可以研究期望的时间点。 <ol><li> 染色细胞准备成像。 <ol><li>以下列终浓度补充染料溶液。 <ol><li>对于基于荧光的分类，在PBS中制备5μg/ mL核染色和5μM死细胞染色（ 参见表材料 ）。 </li><li>对于基于形态学的分类，在PBS中制备5μg/ mL核染色剂，5μM死细胞染色剂和5μM细胞染色剂（ 参见表材料 ）。 </li></ol></li><li>向每个孔中加入20μL染料溶液。在37°C孵育30分钟防光。 </li></ol></li><li> 优化和获取图像。 <ol><li>从培养箱中取出孔板，用70％EtOH擦拭板的底部，并将板放置在成像室中。 </li><li>在"设置"选项卡中，单击"通道选择"下的"+"按钮添加相应的通道图像（ 即明场，核染色，死细胞染色，GFP和RFP信号）。 </li><li>单击"布局选择"，并从0μm开始，以2μm开始，以2μm为单位进行z层叠的测试图像，并以20μm结束，以识别对焦平面。为"高度"下的每个通道输入此距离。 </li><li>点击每个通道下的"快照"来评估强度和优化曝光时间。根据需要在"时间"下输入更高或更低的值。 </li><li>在屏幕的右侧，在板原理图上突出显示适当的孔（待成像）。在下面的井示意图中，以类似的方式突出显示要成像的二十五个场。 </li><li>在"运行实验"下，在左侧选项卡上的"板名称"中标识板，然后单击"开始"按钮（下面），以10X物镜运行图像采集协议，以在上述通道中生成灰度TIFF图像。 注意:分步骤成像协议的拍卖在仪器之间可能不同，但参数值仍应进行优化。 </li><li>在第二天和第三天重复成像。 </li></ol></li></ol>图像分析注意:所有图像分析都是使用专有软件进行的。然而，由于这不是公开的，因此还在CellProfiler 2.2和CellProfiler Analyst 2.0上设计了可比较的分析，下面列出了一个简短的协议，并提供了详细的协议作为补充材料（测试图像已经被加载到管道中以测试工作流程）。来自该实验的图像在每个井基础上分组在一起，使得每个孔可以单独加载。下面的CellProfiler管道包含一个正则表达式，从每个图像文件名分析元数据信息，并允许图像按渠道进一步分组。 <ol><li>下载并安装开源软件:&#39;CellProfiler9;和"CellProfiler Analyst"。在安装期间接受所有默认选项和加载项。 </li><li> 将异源细胞培养物分类为亚群体。 <ol><li>打开"CellProfiler"软件，单击"文件"| "进口管道"| &#39;从文件&#39;，并选择相应的文件（"荧光分类cppipe"进行基于荧光的分类，或"Morphology_Classification.cppipe"用于基于形态的分类）。 注意:这些文件包含基本图像处理和核/细胞分割的协议。由于这些细胞上存在不均匀的Hoechst染色体，细胞核被分割和扩大，然后用于掩蔽图像以允许以更低的强度分割细胞核。 <ol><li>选择基于荧光的分类流程，根据eGFP强度将细胞分类为H3255或CCD-19Lu，并计算形态特征。 注意:CCD-19Lu细胞用GFP转导。 </li><li>选择基于形态的分类管线来细分核和细胞，并计算形态特征。 注意:需要进一步下游分析"CellProfiler Analyst"进行分类。死细胞通过基于荧光的分类进行分类。 </li></ol></li><li>点击屏幕左下角的"查看输出设置"。选择要分析的图像文件的位置（"默认输入文件夹"）和提取数据的目标位置（"默认输出文件夹"）。 </li><li>点击"分析图像"开始分析。分析完成后，点击"确定"，进入"默认输出文件夹"，查看计算的数据。 注: 补充文件1-CellProfilerProtocol.pdf中提供了示例参数值和图像。 </li><li>对于基于形态的分类（仅），请执行以下操作。 <ol><li>打开"CellProfiler Analyst"软件，选择CellProfiler中生成的数据库属性文件，然后单击"打开"。 </li><li>点击屏幕左上角的"分类器"选项卡。要从实验中调用随机单元格图像，请单击"提取";图像将显示在未分类的窗口中。 </li></ol></li><li>通过选择并将细胞拖动到适当的储存箱（见补充文件2-Pipeline.pdf : 图B ），将细胞手动分类为阳性（H3255）或阴性（CCD-19Lu）。每个子群体分类至少50个细胞后，单击"培训分类器"，然后单击"检查进度"。 注意:细胞通过用户监督的随机森林机器学习算法12进行分类 。如果准确度不高于批准阈值（&gt; 90％），可能需要返回并优化"CellProfiler"上的细胞分割，否则细胞可能不适合分类通过形态学。 </li><li>点击"全部评分"为每个子群体生成一个具有细胞计数的表格。 </li></ol></li></ol>

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作者没有什么可以披露的。

The protocol described above improves upon current cell biology assays by providing more comprehensive insights into phenotypic dynamics of multiple cell types in response to environmental perturbations while using reduced reagents and time. A major advantage of this experimental design is the ability to analyze multiple phenotypes with a single setup and generate quantitative data characterizing these phenotypes on a single cell level. One technical advantage to this platform is the ease of initial troubleshooting compared to other assays. Because this method is image-based, one is able to visualize the wells for apparent over/under seeding. It is advisable to have cells in the exponential growth phase for the duration of the experiment and not be limited by nutrient or spatial constraints or confounded by senescence due to scarce seeding. Otherwise, birth and death rates may not be reproducible between experiments. For reference, CellPD is a publicly available program for computation of birth and death rates14. Additionally, one can visualize whether adequate concentrations of dyes were added to each well. Pipetting issues in an individual well could result in missegmentation and skewed data, but can easily be detected with the aforementioned protocol.
Unfortunately, not all cell types may be amenable to this application. It is important to be able to accurately segment the nuclei and cells, therefore analysis of cells that organize in more sphere-like or clumped structures may not be suitable. For some cells, it also may be advantageous to use a cell strainer prior to seeding to ensure initial seeding of single cells. In addition, the linear classifier technique is only applicable to cell types that can be readily distinguished based on morphology features. 
For the success of the protocol, it is important to first optimize the imaging conditions, as the validity of downstream analyses is dependent on the quality of the images. While here we performed experiments using a high-content screening platform, image acquisition can also be performed using any fluorescent microscope (although an automated imaging platform is ideal for high-throughput approaches). Test images should be taken prior to each imaging time point to ensure that there are no problems with the microscope or protocol. The signal to noise ratio should be high, especially for the channels that will be used for segmentation (i.e. nuclei, cell stains). Additionally, it is important to image in the optimal plane of focus. If the images are out of focus, segmentation becomes much more difficult and the calculated morphological features will likely be inaccurate. Illumination differences between fields can cause problems with image segmentation as well. Large differences in brightness make the automated selection of threshold values difficult. Additionally, if there are heterogeneous fluorescence intensities between cells, a single threshold value may not sufficiently segment all the cells in an image. In this analysis, these problems were overcome by creating masks around brighter and dimmer cell populations and segmenting each population separately. 
While the phenotypes under investigation in this protocol are limited to live, dead, and morphological characterization, they can easily be expanded to investigate other features. For example, functional genetic studies can be added with RNAi, overexpression, or other chemical perturbations. 
In this paper, the capabilities of the protocol to measure the response of non-small cell lung cancer cells to erlotinib in the presence and absence of CAFs was demonstrated. However, this is merely one example of the many cell types and microenvironmental parameters that can be tested. We have extended this protocol to be used with other cell types and drug studies, including primary cells isolated from patient tumors13,15.

基于细胞的测定在基础研究和药物开发环境中已经成为一个主力。然而，这些标准测定的局限性随着体外和临床数据的不一致以及大多数药物未能获得FDA批准而变得越来越明显。在这里，我们提出了一种利用定量成像同时分析异源细胞表型以响应相关的共同环境刺激的新方法。 用于测量细胞活力的传统的基于细胞的测定法包括:台盼蓝排除测定，MTT / MTS和Annexin V-FITC流式细胞术染色。台盼蓝排除测定虽然简单而便宜，但需要大量的细胞，这是耗时的，并且经常受到用户偏倚的影响1 。 MTT和MTS测定通过测量线粒体代谢率间接测量细胞活力。然而，代谢活性细胞的数量可能受到不同培养条件（如培养基或氧浓度）的影响，导致不准确的结果，并阻止细胞类型和条件的标准化2,3 。这些技术的另一个主要缺点是它们无法区分多种细胞类型 - 大多数生物系统是异源细胞的。虽然流式细胞术方法具有区分多细胞群体的能力，但需要细胞标记，动态取样具有挑战性，而当使用贴壁细胞时，该应用程序变得耗时且容易出错。 其他重要的细胞表型，包括形态学变化，发生在响应于环境刺激，但不被传统的基于细胞的测定法捕获。通过形态表征和映射样本之间的相似性，分析细胞状态是一个强大的，无偏见的工具能够提供基础和翻译研究的许多方面的新见解，包括基础细胞生物学和药物发现4 。此外，已经显示肿瘤细胞形态与肿瘤亚型5和侵袭性6相关 。因此，研究这些细胞特征以及它们如何与特定的环境扰动有关是非常有意义的。此外，人们可以使用形态特征的差异来区分共培养系统中的亚群体。荧光标记细胞具有垮台（ 即改变固有的细胞特性，耗时），因此分类细胞类型的其它方法是有利的。 基于显微镜的成像是以多重，定量和鲁棒的方式分析细胞表型的替代方法。在这份手稿中，我们运用定量成像管道来突出演变肿瘤内异质细胞群的动态变化。我们专注于非小细胞肺癌（NSCLC）细胞和肿瘤相关成纤维细胞（CAF）之间的相互作用，这是肿瘤中最常见的基质细胞类型。 CAFs参与了肿瘤的起始，进展和治疗反应;因此，在不存在CAF的情况下对肿瘤细胞进行表型测定可能会误导7,8,9 。具体来说，我们评估了CAF对肿瘤细胞对厄洛替尼的影响，厄洛替尼是靶向经常用于NSCLC临床治疗的表皮生长因子受体（EGFR）的小分子。我们利用高分辨率的筛选平台及其附带的图像分析软件进行评估;然而，为了使其他研究人员可以使用这种方法，我们还使用开源软件开发了可比较的下游协议:CellProfiler 10和CellProfiler Analyst 11 。大多数基于图像的高含量筛选测定用特定于给定仪器模型的商业化软件进行分析。结果很难在其他实验室用不同的软件复制，因为底层算法通常是专有的。使用这种基于图像的管道，测量了使用基于荧光和形态的分类来响应药物治疗的异源细胞培养物的每个亚群的细胞增殖，死亡和形态。以下协议提供了一种鲁棒的方法来探测复杂的蜂窝过程。

<table border="0" cellpadding="0" cellspacing="0" width="677">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">RPMI-1640</td>
			<td style="width:127px;">Corning</td>
			<td style="width:119px;">10-040-CV</td>
			<td style="width:191px;">cell culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">FBS</td>
			<td style="width:127px;">Gemini</td>
			<td style="width:119px;">100-106</td>
			<td>medium supplement</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">Penicillin/streptomycin</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:119px;">15-140-122</td>
			<td>medium supplement</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">TC20</td>
			<td style="width:127px;">Biorad</td>
			<td style="width:119px;">1450102</td>
			<td>cell counter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">TC20 slides with trypan blue</td>
			<td style="width:127px;">Biorad</td>
			<td style="width:119px;">1450003</td>
			<td>cell counter slides</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">96-well plates</td>
			<td style="width:127px;">Corning</td>
			<td style="width:119px;">3904</td>
			<td>clear bottom black plates</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">Erlotinib</td>
			<td style="width:127px;">LC Laboratories</td>
			<td style="width:119px;">E-4007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">DMSO</td>
			<td style="width:127px;">VWR</td>
			<td style="width:119px;">317275-100ML</td>
			<td>solution to resuspend drug</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">Hoechst</td>
			<td style="width:127px;">Invitrogen</td>
			<td style="width:119px;">H21491</td>
			<td>nuclear dye</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">Propidium Iodide</td>
			<td style="width:127px;">Invitrogen</td>
			<td style="width:119px;">P1304MP</td>
			<td>dead cell stain</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:240px;">Cell Tracker Orange CMRA</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;">C34551</td>
			<td>whole cell stain</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:240px;">Operetta high content imaging system</td>
			<td style="width:127px;">Perkin Elmer</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:240px;">CellProfiler</td>
			<td style="width:127px;">Broad Institue</td>
			<td style="width:119px;">version 2.2.0 (rev 9969f42)</td>
			<td>http://cellprofiler.org/releases/</td>
		</tr>
		<tr height="86">
			<td height="86" style="height:86px;width:240px;">Cell culture incubator</td>
			<td></td>
			<td></td>
			<td style="width:191px;">Any cell culture incubator will be suitable - cells were cultured under 37 &#186;C at 5% CO2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">15 mL Falcon conical tubes</td>
			<td style="width:127px;">Falcon</td>
			<td style="width:119px;">14-959-53A</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:240px;">10 cm2 cell culture plates</td>
			<td style="width:127px;">TPP</td>
			<td>93040</td>
			<td></td>
		</tr>
	</tbody>
</table>

discrimination and characterization of heterocellular populations using quantitative imaging techniques

细胞过程是复杂的，并且是由多种细胞类型与其环境之间的相互作用产生的。现有的细胞生物学技术往往不能准确地解释这种相互作用。使用基于定量成像的方法，我们提出了一个高含量的方案来表征异质细胞群体对环境刺激变化的动态表型反应（ 即形态学变化，增殖，凋亡）。我们强调我们根据应用来区分基于荧光强度或固有形态特征的细胞类型的能力。该平台允许更全面地表征亚群对扰动的反应，同时利用更短的时间，更少量的试剂，并且具有比传统细胞生物学测定更低的误差可能性。然而，在一些情况下，细胞群体可能难以基于复合纤维素进行鉴定和定量lar功能，需要额外的故障排除;我们强调协议中的某些情况。我们在癌症模型中使用对药物的反应来证明这一应用;然而，它可以更容易地应用于其他生理过程。该协议允许人们识别共培养系统内的亚群体，并表征每个人对外部刺激的特定反应。

我们生成了一个由25个字段/孔，54个孔/板（3个细胞群体×6个药物浓度×3个重复）组成的图像组，跨越三个平板，共有4,050个单独的图像。在实验过程中生成的图像集使用专有软件（参见材料表）进行分析，以提取细胞的各种定量性质（ 即形态学，荧光），然后可用于分类细胞亚群。然而，由于所使用的商业软件的访问有限，因此创建了CellProfiler和CellProfiler Analyst中可比较的下游管道。 异种细胞分类为亚群基于DNA染色（这里是Hoechst）鉴定和分割细胞核，并且基于荧光或莫尔将细胞群分类形态学（ 图1 ）。对于基于荧光的分类，成纤维细胞（CCD-19Lu）预先用GFP-慢病毒转导。测量每个核的GFP强度水平，并且计算高于允许阈值（基于背景信号）的那些被分类为CCD-19Lu，而下面被鉴定为肿瘤细胞（H3255）。对于基于形态学的分类，细胞先前用无毒细胞染色剂染色（参见材料表），并将其用于鉴定和分割细胞质。训练每个人口约50-100个细胞的机器学习算法。鉴定出种群间的形态特征差异显着，然后将其用于设计线性分类器以区分CCD-19Lu和H3255细胞。荧光和形态分类方案在区分两个细胞群体时一致为97.4％（n = 1403）在药物治疗条件（1μM埃罗替尼）中，92.5％（n = 916）与未治疗条件相同（ 图2 ）。 子群体表型分析除了区分细胞类型之外，我们旨在表征每个亚群的表型性质。多重测试可节省时间和试剂，增加一致性，并提供有关正在研究的系统的其他信息。有许多潜在的表型产出，应根据感兴趣的问题选择它们。在这里，研究了响应于厄洛替尼治疗的细胞形态和活力状态的变化。经过三天的药物治疗后，观察到H3255细胞的核面积减少和细胞面积的增加（ 图3A ）。核区域之间的平均差异通过双侧型-2（相等方差） t检验（ p = 7.92×10 -16 ），发现"无药物"和"药物"治疗的人群具有统计学显着性。我们假设这种观察是对药物治疗施加的压力的细胞反应。 研究药物是否具有细胞毒性（ 即 ，随着时间的推移死亡细胞数量的增加）或细胞抑制（ 即随着时间的推移而减少细胞出生数）对细胞的影响，因为这具有深刻的临床影响。例如，细胞抑制药物作用诱导生长停滞，但不能消除肿瘤中的细胞，因此一旦药物被除去，癌细胞就有可能重新启动细胞增殖。药物作用常常是上下文，浓度和细胞类型依赖性。我们以前观察到厄洛替尼在一种细胞类型中引发细胞毒性反应，同时显示ac在另一个13的 ytostatic反应。 传统的活力测定法输出相对细胞数，因此，不区分生长停滞和细胞死亡。在此，基于碘化丙啶染色鉴定死细胞（ 图3B ）。观察到厄洛替尼对H3255细胞的细胞毒性和细胞生长抑制作用，药物治疗后死亡人数增多，出生人数减少（ 图3C ）。值得注意的是，由于细胞清除，死亡细胞数量在第1天后下降。 CCD-19Lu细胞不受药物的影响。该平台的另外一个优点就是生成定量数据。例如，在我们的共培养实验中，在没有或与厄洛替丁组合的三天后，发现1,118（75.8％）H3255细胞的初始亚群为2,817（87.9％）或396（57.2％）ib处理（ 图4 ）。因为我们可以产生实际细胞计数而不是相对百分比（如流式细胞术方法），我们得出结论，药物治疗期间组成的变化是由于H3255细胞减少而不是CCD-19Lu的增加。由于细胞清除，死亡率可能被低估是值得的，这是难以通过实验评估，并且可能在细胞类型之间不同。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55844/55844fig1.jpg" /> 图1:图像分析协议概述。两个潜在的下游图像分析管线可以使用基于形态的分类或基于荧光的分类对异源细胞群进行分类。刻度棒=100μm。 <a href="http://ecsource.jove.com/files/ftp_upload/55844/55844fig1large.jpg" target="_blank">请点击她</a>e查看这个数字的较大版本。 <img alt="图2" class="xfigimg" src="/files/ftp_upload/55844/55844fig2.jpg" /> 图2:形态与基于荧光的分类之间的一致性。 （ A ）显示两个分类协议重叠的一致图。使用基于形态和荧光的分类将相同的细胞分类为H3255。两种方案是一致的，分类为未处理细胞的97.4％（n = 1403）和用厄洛替尼处理的细胞的92.5％（n = 916）（注意:白色区域太小而不可视化）。 （ B ）10X图像描绘了基于荧光和基于形态的分类之间良好和差的一致性的示例。白色箭头指出在平台之间不一致的细胞。输入图像:蓝 - 核（Hoechst）;绿色 - CCD19Lu（GFP）。 CLASSI化妆图像:红色 - H3255;绿色 - CCD-19Lu。刻度棒=100μm。 <a href="http://ecsource.jove.com/files/ftp_upload/55844/55844fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55844/55844fig3.jpg" /> 图3:单个实验设置的复用表型测量。 （ A ）在存在和不存在药物时，在单细胞水平上计算细胞核和细胞面积等形态特征。注意:测量小于100μm2的细胞区域被认为是碎片，并排除在分析之外。箱体图描绘了具有第一和第三四分位数范围和95％置信区间误差条的中值。 （ B ）共同培养H3255（蓝色）和CCD-19Lu（绿色）细胞，基于丙酸的强度鉴定死细胞碘化镉染色（红色），并使用10X物镜成像。刻度棒= 1 mm（顶面）; 100μm（底部图像）。 （ C ）用或不用药物治疗三天计算活细胞死亡细胞数，活细胞数量明显减少，加上厄洛替尼死亡细胞数量增加。误差条代表基于三次重复的平均值的标准误差。 <a href="http://ecsource.jove.com/files/ftp_upload/55844/55844fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55844/55844fig4.jpg" /> 图4:随时间推移的亚群动态。在含有和不含药物的第0天或第3天，含有H3255（蓝色）和CCD-19Lu（绿色）的孔的代表性10X图像。计数属于每个亚群的细胞，比例饼图显示a样本中人口组成的变化趋势。刻度棒= 1毫米（中间面板，闪烁;最后面板），1毫米（顶部图像），100μm（底部图像）。 <a href="http://ecsource.jove.com/files/ftp_upload/55844/55844fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques at JoVE.com

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

我们已经开发了一种用于研究异源细胞群体动力学以响应扰动的新方案。该手稿描述了基于成像的平台，其产生定量数据集，以稳健的方式同时表征异源细胞群体的多种细胞表型。

使用定量成像技术鉴定和表征异源细胞群体

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do ...

discrimination-characterization-heterocellular-populations-using

Universidad San Sebastian

Research

JoVE Journal

Biology

7.3K 次观看.  University of Southern California (USC). 我们已经开发了一种用于研究异源细胞群体动力学以响应扰动的新方案。该手稿描述了基于成像的平台，其产生定量数据集，以稳健的方式同时表征异源细胞群体的多种细胞表型。 

视频: Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

科研

生物学

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

技术成像钙 2 +信号在人类精子

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

使用具体远藤和exoglycosidases糖基化蛋白质的鉴定和表征

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

采用先进的3D荧光显微镜定量分析自噬

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

测量细胞内Ca<sup&gt; +</sup在人类精子的变化采用4项技术:传统的荧光，停流荧光，流式细胞术及单细胞成像

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

对于外小泡的使用流式细胞仪分析技术

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

发展与表征<em&gt;体外</em&gt;微血管网络和内皮的定量测量的[Ca<sup&gt; 2+</sup&gt;]<sub&gt;我</sub&gt;和一氧化氮含量

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

成像前中期和减数分裂中期的我固定技术<em&gt;果蝇</em&gt;卵母细胞

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

钙化事件的表征在海洋Tubeworm使用实时光学和电子显微镜技术

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...