JB是CRUK临床研究员，AS是一个威康信托基金会博士研究生，在SR实验室是由CRUK高级研究奖学金（C99667/A12918）资助。这项工作是由两个威康信托基金会战略奖（097945/B/11/Z和095931/Z/11/Z）的支持。

1，细胞培养及处理<ol><li>收集在表1中，以培养人骨肉瘤（U2OS）细胞中列出的试剂和设备。准备好所有试剂，并对所有组织培养技术在空气层流罩，以保证无菌</li><li>制备培养基如下:加入50 mL的FCS，加入5ml L-谷氨酰胺和5 ml青霉素/链霉素的DMEM中，以达到最终浓度为10％（FCS），2mM的（L-谷氨酰胺），50单位/毫升（青霉素）和50单位/毫升（链霉素）。温热的培养基中，在37℃的水浴中。 </li><li>准备细胞治疗: <ol><li>几种消毒盖玻片的70％乙醇，在空气中晾干，然后将一个在6个3.5 CM平板的底部。 </li><li>沉浸在2毫升的盖玻片回暖完全DMEM（37℃），按下与盖玻片钳，以确保它们保持附着在孔的底部。 </li><li>从他们的邪教组织分离U2OS细胞URE板通过用3ml PBS洗涤一次，施加4毫升温热0.05％胰蛋白酶-EDTA（1％），并在37℃温育7分钟。 </li><li>重悬细胞6毫升温热的完全DMEM以灭活胰蛋白酶-EDTA，并使用血细胞计数器计数。 </li><li>板2×10 5细胞以每点1.3.1编制的3.5厘米板。轻轻摇动平板，以确保均匀的细胞分布。使细胞附着过夜处理之前。 </li></ol></li><li>通过将4个3.5 CM平板进入缺氧工作站处理细胞并保留其他两个3.5 CM平板在培养箱中37℃，正常氧条件。准备好开始治疗的条件下，RNA标记法（在缺氧室缺氧处理的细胞）1小时的缺氧治疗结束前。 </li><li>暴露的细胞对缺氧的1小时15分钟，1小时30分钟，2小时和24小时。这些时间点是灵活的，用户确定的。 </li><li>使用无论正血球XIC3.5厘米片作为阳性对照，用放线菌素D（10μg/ ml的终浓度）为4小时，作为阴性对照处理的含氧量正常3.5厘米板。放线菌素D是全球RNA合成的抑制剂。 </li></ol>注意事项: 赫斯特荧光染料33342是一种已知的诱变剂。 DMSO是已知的，以促进有机分子进入组织中。 氢氧化钠有腐蚀性。 盐酸具有腐蚀性。 多聚甲醛是所有动物剧毒，可致人类死亡。 下面直接接触的Triton X-100可引起皮肤过敏。 放线菌素D是有毒的皮肤或吞食接触时。 采取适当的预防措施处理所有危险化学品时。 2，点击-IT分析<ol><li>收集下列试剂所需要除了按此-IT试剂盒:磷酸盐缓冲盐水（PBS）pH值7.2 - 7.6，3.7％多聚甲醛（PFA）的PBS，1％的Triton X在PBS中的-100，去离子水（DH 2 O），二甲基亚砜（DMSO），10μM氢氧化钠（NaOH），和37％的盐酸（HCl）;可选 - pH值试纸。 </li><li>进行点击，它的反应如下之前准备一个新鲜的PFA原液: <ol><li>把1.85克PFA，3.5毫升DH 2 O和10微升16:05 M氢氧化钠到50毫升猎鹰管。煮沸300 - 400毫升H 2 O的在一个大烧杯中的微波，使水浴并在通风橱受不了这个。 </li><li>放置50毫升隼入水浴中，并轻轻搅拌10分钟后，周期性地释放所述盖子直至PFA溶解。 </li><li>通过0.2微米的过滤器的注射器的PFA溶液到另一个50毫升Falcon管中，产生了37％的溶液。 </li><li>通过加入PBS稀释10倍的这个和通过加入约12微升的浓盐酸使pH为6.8。选项​​:确认正确的pH值在通风柜的试​​纸。 </li><li>立即使用或存放Øvernight在4℃下</li></ol></li><li>制备储备溶液的核糖核酸成像试剂盒如下: <ol><li>加入373微升DH 2 O至导致欧盟100毫米的原液成分A。储存在-20℃下长达一个月。 </li><li>加85微升的DMSO中，以组分B（的Alexa Fluor 594）和通过移液或涡旋混匀。无论是在-20℃可保存一年储存。 </li><li>加入2ml DH 2 O至E成分以创建RNA成像试剂盒反应缓冲液添加剂的10X的解决方案。储存在-20℃下长达一年，丢弃时的解决方案开发了一个棕色的颜色。 </li></ol></li><li>细胞与欧盟标签:准备欧盟从预热的完全培养基（37℃）制备步骤2.3的100毫米的股票2X工作液执行此步骤的余下并且在缺氧室步骤2.5与低氧处理的细胞 。 ，通过添加本2X工作溶液等体积的介质续稀释至1X癌宁细胞。孵育治疗的细胞培养条件下1小时。 </li><li>细胞固定:每洗一次很好用PBS加1毫升3.7％的煤灰股票准备在上面的步骤2.2处理条件下。孵育处理条件下15分钟。 与低氧处理后的样品可以从缺氧室中在这一点上被移除 。去除固定液，用PBS洗涤每孔一次（ 请小心处理煤灰的浪费负责 ）。执行下面的步骤在室温下。 </li><li>通透:取出洗涤溶液并加入1 ml 1％的Triton X-100的PBS中的每个孔中。孵育15分钟，在室温下进行。除去透化缓冲液并用PBS洗涤各孔一次。 </li><li>标记的RNA检测: <ol><li>在DH 2 O稀释10X溶液（步骤2.3准备）1:10准备的RNA成像试剂盒反应缓冲液添加剂的新鲜的1X工作液</li><li>准备RNA成像套件配制后立即按表2使用的反应鸡尾酒。 </li><li>去除洗涤液，并加入500μl的RNA成像试剂盒反应鸡尾酒到每个样品。孵育30分钟，在室温， 避光 。 </li><li>除去核糖核酸成像试剂盒反应混合物和用1ml RNA成像试剂盒反应漂洗缓冲液（F成分）洗一次，然后拆下核糖核酸成像试剂盒反应漂洗缓冲液。 </li></ol></li><li>可选多重反应:在这一点上，根据制造商的建议进行样品的额外抗体标记。 </li><li> DNA的染色:洗涤样品，用PBS然后除去洗涤液。稀释的Hoechst 33342（G成分）1:1,000在PBS。加入1毫升稀释的Hoechst 33342至每个孔，并孵育15分钟，在室温下， 避光 。取出的Hoechst 33342溶液并用PBS洗细胞两次。取出洗净solution和进行光镜。 </li></ol> 3，光学显微镜<ol><li>摩盖玻片: <ol><li>把10微升在标准显微镜载玻片的中心安装的介质。 </li><li>将盖玻片与细胞侧朝下放在显微镜载片上的中心以覆盖安装介质的等分试样。注意避免在安装介质中气泡的产生，因为这会掩盖后续的图像采集。 </li><li>通过应用明确的指甲油，以它的周长坚持盖玻片在显微镜幻灯片。允许在室温下干燥5分钟。 避光保存 。样品可以贮存于-20℃下以下安装。 </li></ol></li><li>图像采集:采用宽视场显微镜能够荧光检测获取图像。为的Alexa Fluor染料和赫斯特荧光染料33342绑定到DNA的近似荧光/激发发射最大值示于表3中 。 </li><li> PERFOR使用40X/1.30 NA油浸镜头和拍摄的影像具有冷却CCD相机米影像。 </li></ol> 4，数据分析<ol><li>使用图像处理软件（见材料表）导入到客户端OMERO之前去卷积图像。正常化通过施加从控制氧正常细胞中的渲染设置到所有其他条件呈现在相同的实验中，显微镜图像可视化和分析的软件设置为所有图像。 </li><li>使用感兴趣区域（ROI）工具在显微镜图像可视化和分析软件（见材料表），以从每个图像选择核。获得平均强度为每个投资回报率，并通过选择强度分析选件，其中包括一批30-50单元格之间计算每个条件的平均值和标准偏差。 </li><li>通过绘制步骤4.2吨获得的所有个体的平均亮度值使用数据绘图软件（见材料表）构建散点图Ø反映各条件下的细胞之间的差异。或者构建条形图来表示平均平均强度加上新生RNA标准偏差。 </li></ol>

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JRS是方正伦科软件公司，一个开源的总部设在美国的商业公司，有助于OMERO的。

我们已经描述了我们使用的RNA成像套件的研究缺氧对RNA合成的骨肉瘤（U2OS）细胞的作用。这一技术相对比较简单，并提供了有关在单细胞水平全局转录数据。我们修改了传统的协议，这个套件包含标签在缺氧室。据我们所知，这是使用这种技术来测量缺氧改变RNA合成的第一份报告。我们所使用的RNA的成像试剂盒适用于实验中缺氧，因为它不需要放射性同位素，并与在受控气氛工作站协同工作的限制技术上不兼容。常见的问题该技术关注有效固定和样品的标记。这是极为重要的是，煤灰用于固定样品是新鲜的和描述的一样，以确保样品的低背景染色执行遵循&#39;点击&#39;反应，该洗涤步骤。若bACKGROUND荧光变得建议的步骤2.7.4后洗一次，用1ml的RNA成像试剂盒反应漂洗缓冲的一个问题。 这里提到的RNA成像套件代表了RNA成像技术一个显著进步在易用性和特异性的反应和速度方面。这种技术是通过它需要以标记样品中的时间主要限制。 RNA的成像套件需要，防止在全球RNA的快速变化通常会发生此期限内的考试有1小时的贴标时间。正是由于这个原因，我们的第一时间点被限制为1小时和15分钟。该技术被用在很大程度上涉及与试剂的相容性和其它荧光标记物，例如，该RNA成像试剂盒不与鬼笔环肽染色兼容其他一些局限性有关。 研究RNA合成细胞的所有现有的方法都是基于修饰核苷的掺入新生RNA和结合标签通过不同方式的检测。开拓方法是基于用放射性标记的RNA前体随后检测与放射自显影。这种方法导致了发现细胞周期阶段和其他重要发现;然而，它有很多缺点，例如放射性工作中，长曝光时间和放射自显影6的低分辨率图像的笨重的性质。在该领域的未来提前利用免疫组织化学的方法，以消除放射性的必要性。 RNA的标记由BRU注册成立后免疫组化检测。然而，有效的抗体结合所需核酸变性，改善获得的DNA或导致的细胞形态丢失和损坏的许多蛋白质的抗原决定簇，以阻止其进一步的检测与荧光标记的抗体13的RNA。的"点击化学"技术的引入简化了检测步骤和t他的程序相比，放射自显影和免疫组化。该技术是基于叠氮 - 炔胡伊斯根环加成反应，其中的5-ethyniluridine末端炔烃与叠氮化物与结合在铜（I）的存在荧光团结合。该反应是特异性，快速，不需要任何额外的步骤，并且与免​​疫化学检测其它细胞成分容易相容。 使用该RNA的成像技术，我们发现，细胞，在相同的单层人群中，有不同的RNA合成水平响应于缺氧。我们限制了我们的实验中只检查RNA生产的影响;但是，它是完全可以用这样的RNA成像试剂盒进行修改，附加的多重反应中，允许对RNA的产量的更复杂的分析。例如，使用特异于活跃转录的标记物，如磷酸化的RNA聚合酶II或transla的偶数分量电平的次级抗体染色化机械给这个新产生的RNA被转化成蛋白质的量的指示是可能的。额外的蛋白质，如肌动蛋白，这不应该与低氧显著改变的蛋白质，可以测试作为一个额外的控制。此外，不同类型的细胞可以被测试，因为这很可能是不同的单元将具有不同的RNA合成速率和甚至不同的反应缺氧。 使用该RNA成像试剂盒是相当简单的提供作为所述被执行的步骤。在协议中的关键步骤涉及细胞接种，细胞固定和染色和图像采集。该板块的细胞所描述的密度，防止过或正在汇合在实验这将显著影响的结果是很重要的。同样重要的是用新鲜固定液，保证了电池的取最好的"快照"，并染色以减少BAC后洗涤细胞时要小心kground到一定水平，是可以接受的效率分析。最后，图像采集的方法是实现这一目标是一个质量足够好，以供后续分析的图像非常重要。我们建议您使用的最佳光镜可检查样品光学如本协议，可在最密集的研究中心在全球范围使用的显微镜​​。

当正常的氧气供应，组织受到干扰缺氧（低氧张力）发生。环境氧既是养分和信号传导分子，提供了重要的线索对许多类型的细胞。被改变的环境氧气通过一组控制的重要转录因子家族被称为缺氧诱导因子（HIFs）的双加氧酶的活性的检测。该HIFs是由两个亚基组成，α和β。有三种已知的（3 1，2，和）同种型，HIF-α和HIF-1β的多个剪接变体。 HIF-1β组成型表达，而不是由环境的氧气水平1调节。缺氧诱导因子-α家族成员是由一类脯氨酰羟化酶（博士）动态地调节和抑制因子缺氧诱导因子（FIH）;两者都需要氧作为辅因子催化HIF-α2,3的羟化。常氧中的HIF-α家族成员羟基化和标记为proteosom人降解的E3连接酶，希佩尔林道（VHL）。在缺氧的博士和富士康是无效的或具有活性降低。的HIF-α亚型变得稳定时，形成与HIF-1β的异源二聚体，以及影响基因，提供对低氧环境（ 图1A）的细胞应答的转录4。 当前技术用于RNA分析集中于平均值的跨越给定的细胞群体的定量。细胞对缺氧的刺激所引发的基因，使他们能够适应他们的恶劣环境5无数的转录反应。然而，缺氧往往存在一个梯度，以及细胞在缺氧的环境不受统一的缺氧刺激。我们描述了按此-IT RNA成像试剂盒的实施方案中的控制氧工作站检查全局RNA合成在缺氧单细胞水平。 RNA的成像试剂盒采用一lkyne-修饰核苷，5 -乙炔基尿嘧啶核苷（EU）和化学选择性连接，能够检测全局的RNA合成的时间和空间中的细胞和组织6。简言之，将细胞用缺氧和在欧盟的存在下进行培养。然后，他们的固定和通透和欧盟纳入新生RNA是由欧盟的化学选择性结扎用含叠氮染料的检测。一个典型的工作流用于此反应示于图1B中 。我们所使用的RNA的试剂盒的成像检查RNA的合成在起因于缺氧处理单细胞水平。 炔标签的小尺寸使得修饰的核苷的有效掺入到RNA的特异性。的化学选择性结扎或&#39;点击&#39;的反应是高效，快速和具体7-10。所有的反应成分是生物惰性，反应不需要极端的温度或溶剂。点击反应否定要求对传统的放射性标记，并允许结果的直接可视化，因为输出是光。另外，检测分子可以很容易地穿透复杂样品，允许多重分析包括抗体用于RNA-相互作用蛋白的检测。这种RNA成像法是用有机染料包括的Alexa Fluor和荧光素（FITC）兼容。 我们测量的RNA合成的变化下使用开放式显微镜环境下对远程对象（OMERO）的实施治疗我们的细胞。 OMERO是开源软件，可以在<a href="http://openmicroscopy.org/">http://openmicroscopy.org/</a> 。这种显微镜图像可视化和分析软件可以访问和使用范围广泛的生物学数据，包括多维，异构数据集的管理。客户端应用程序允许远程可视化和复杂的生物图像数据11的分析;我们用它来量化在全球和单细胞RNA合成的视觉变化。使用该显微镜图像可视化和分析软件这些数据，并分析了RNA的成像实验所需的步骤如下所示。 我们看着下面的人骨肉瘤的治疗（U2OS）细胞缺氧长达24小时的全球RNA合成的变化。在所有情况下，我们检测到细胞 - 细胞间的变化的RNA生产水平。低氧暴露短的时间内未产生显著改变在细胞新生RNA的水平。然而，暴露于缺氧24小时导致RNA的细胞产生的量显著增加。大多数细胞对低氧的是以下的曝光时间较长，例如4〜24小时来测定。然而，一些机制落实到位早得多，例如; NF-κB的激活发生在低氧暴露12的5-15分钟。调查较短的低因此霞曝光时间是必要的，并可能有损于更复杂的反应，如细胞周期和凋亡。

<table border="0" cellpadding="0" cellspacing="0" style="width:1313px;" width="1314">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:236px;">U-2 OS Primary Cells</td>
			<td style="width:92px;">European Collection of Cell Cultures</td>
			<td align="right" style="width:115px;">92022711</td>
			<td style="width:168px;"></td>
			<td style="width:703px;"><a href="http://www.hpacultures.org.uk/products/celllines/generalcell/search.jsp?searchtext=u2os&amp;dosearch=true">http://www.hpacultures.org.uk/products/celllines/generalcell/search.jsp?searchtext=u2os&#38;dosearch=true</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Gibco</td>
			<td align="right">14190094</td>
			<td></td>
			<td><a href="http://products.invitrogen.com/ivgn/product/14190094">http://products.invitrogen.com/ivgn/product/14190094</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Gibco</td>
			<td align="right">41966029</td>
			<td></td>
			<td><a href="https://products.invitrogen.com/ivgn/product/41966029?ICID=search-41966029">https://products.invitrogen.com/ivgn/product/41966029?ICID=search-41966029</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FCS</td>
			<td>Gibco</td>
			<td align="right">10270106</td>
			<td></td>
			<td><a href="http://products.invitrogen.com/ivgn/product/10270106?ICID=search-10270106">http://products.invitrogen.com/ivgn/product/10270106?ICID=search-10270106</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin</td>
			<td>Lonza</td>
			<td>DE17-603E</td>
			<td></td>
			<td><a href="http://www.lonza.com/products-services/bio-research/cell-culture-products/reagents/antibiotics-antimycotics/penicillin-streptomycin.aspx">http://www.lonza.com/products-services/bio-research/cell-culture-products/reagents/antibiotics-antimycotics/penicillin-streptomycin.aspx</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>Lonza</td>
			<td>BE17-605E</td>
			<td></td>
			<td><a href="http://www.lonza.com/products-services/bio-research/cell-culture-products/reagents/cell-culture-reagents/l-glutamine-200mm.aspx">http://www.lonza.com/products-services/bio-research/cell-culture-products/reagents/cell-culture-reagents/l-glutamine-200mm.aspx</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.05% Trypsin-EDTA (1%)</td>
			<td>Gibco</td>
			<td>25300062</td>
			<td></td>
			<td><a href="http://products.invitrogen.com/ivgn/product/25300062">http://products.invitrogen.com/ivgn/product/25300062</a></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Hypoxia Chamber</td>
			<td>Ruskinn</td>
			<td>InVivo2 300</td>
			<td></td>
			<td><a href="http://www.ruskinn.com/products/invivo2300">http://www.ruskinn.com/products/invivo2300</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Click-iT assay Kit</td>
			<td>Invitrogen</td>
			<td>C-10329/C10330</td>
			<td></td>
			<td>http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catDisplayStyle&#38;catKey=601107&#38;filterDispName=Nascent%2BRNA%2BDetection%2B%2526amp%253B%2BCapture%2BKits&#38;_bcs_=H4sIAAAAAAAAAMWP3WrDMAyFn8Y3MwtOUrrcZg0dpVAGZbs3jpYIEjvYSoLffvK6jbKV3Q6EhP44%0A37nPhaqevWtnQ0GKYivP4Bc0EP6Y90STKGtR7DnWdc3QLkjedWAz40YeBiTgMgdOYDn1boSsp3Hg%0Ad1GUKVRFfobUqwfFJVc5H1aFyu%2B4O%2BJFV48s9SjrEHT8pT19Av4EKOtK8RqXzn4BvJw56RY9GEqr%0A7wdR7s3YirI5vD6djKYGwzTouNMEnfOREXh4hMgXie2a%2F00P4aYBLsV2czFyms0AaGRtsJUNEOuj%0As9fWrB5iwCCT5X92%2BEF9y%2BI7x9UoYCgCAAA%3D</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pFA</td>
			<td>Sigma</td>
			<td>76240</td>
			<td></td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/fluka/76240?lang=en&amp;region=GB">http://www.sigmaaldrich.com/catalog/product/fluka/76240?lang=en&#38;region=GB</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma</td>
			<td>X-100</td>
			<td></td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/x100?lang=en&amp;region=GB">http://www.sigmaaldrich.com/catalog/product/sial/x100?lang=en&#38;region=GB</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10M NaOH</td>
			<td>Sigma</td>
			<td>72068</td>
			<td></td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/72068?lang=en&amp;region=GB">http://www.sigmaaldrich.com/catalog/product/sigma/72068?lang=en&#38;region=GB</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">37% HCl</td>
			<td>VWR</td>
			<td>20252.335</td>
			<td></td>
			<td><a href="https://uk.vwr.com/app/search/Search?keywords=%2720252.244%27%20%2720252.290%27%20%2720252.324%27%20%2720252.335%27%20%2720252.368%27%20%2720252.420%27%20%2720252.448%27%20%2720252.980%27&amp;searchOp=or">https://uk.vwr.com/app/search/Search?keywords=%2720252.244%27%20%2720252.290%27%20%2720252.324%27%20%2720252.335%27%20%2720252.368%27%20%2720252.420%27%20%2720252.448%27%20%2720252.980%27&#38;searchOp=or</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VectaShield Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1400</td>
			<td></td>
			<td><a href="http://www.vectorlabs.com/catalog.aspx?prodID=1483">http://www.vectorlabs.com/catalog.aspx?prodID=1483</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Actinomycin D</td>
			<td>Sigma</td>
			<td>A9415</td>
			<td></td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a9415?lang=en&amp;region=GB">http://www.sigmaaldrich.com/catalog/product/sigma/a9415?lang=en&#38;region=GB</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Deltavision Core Microscope</td>
			<td>Applied Precision</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.appliedprecision.com/">http://www.appliedprecision.com/</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">softWoRx [Refered to in text as image processing software]</td>
			<td>Applied Precision</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.api.com/softworx.asp">http://www.api.com/softworx.asp</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CoolSnap HQ2 CCD Camera</td>
			<td>Photometrics</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.photometrics.com/products/ccdcams/coolsnap_hq2.php">http://www.photometrics.com/products/ccdcams/coolsnap_hq2.php</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Open Microscopy Environment for Remote Objects (OMERO) [refered to in text as microscope image visualisation and analysis software]</td>
			<td>OME</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://openmicroscopy.org/">http://openmicroscopy.org/</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SigmaPlot v12.0 [Refered to in text as data graphing software]</td>
			<td>Systat Software Inc.</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.sigmaplot.co.uk/products/sigmaplot/sigmaplot-details.php">http://www.sigmaplot.co.uk/products/sigmaplot/sigmaplot-details.php</a></td>
		</tr>
	</tbody>
</table>

analysis of global rna synthesis at the single cell level following hypoxia

的氧气供应缺氧或降低参与了许多生理和病理过程。在分子水平，细胞启动一个特定的转录程序，以安装适当的和协调一致的细胞反应。该电池具有需要分子氧作为辅助因子的活性几个氧传感器的酶。这些范围从脯氨酰羟化酶组蛋白去甲基化酶。大多数研究分析细胞对缺氧是基于蜂窝人口和平均的研究，并作为缺氧细胞的这种单细胞分析很少执行。这里我们描述了在低氧单细胞水平通过使用按此-IT RNA的成像盒中的控制氧工作站，随后显微镜分析和定量全球RNA合成的分析方法。采用低氧不同的时间长度的癌细胞，RNA标记和测量的每个小区中。这种分析可以的时间和细胞 - 细胞在全球RNA合成下列低氧应激的变化的可视化。

RNA的成像试剂盒反应的原理图如图1B所示。我们利用这个反应来定量测定总RNA的合成在U2OS细胞在与缺氧处理两个个体（单细胞）和全球层面（细胞群）上。将细胞用缺氧和在欧盟的存在下进行培养。然后，他们被固定和通透和欧盟的成立是由欧盟的化学选择性结扎用含叠氮染料的检测。这"咔哒"反应导致可检测并使用荧光光学显微镜定量的荧光发射。 图2A示出从这个实验的典型结果。荧光标记的细胞被伪彩色红色和图像已在公开显微镜环境对远程对象（OMERO）被打开。这种显微镜图像可视化和分析软件有一个内置的ROI工具，它可用于选择各个亚细胞结构Tures的和定量分析所选区域内的图像强度。数据输出后，在客户端程序中使用这个工具的一个典型例子可以看出，在图2B;单个电池强度显示从旁边用作目视检查，以确保数据的有效性实验的代表图像。 单个细胞的荧光强度，可以绘制为散点图来显示个人的蜂窝强度的处理过的细胞群体中的分布。这种类型的曲线图的一个例子是出现在图2C中 。因为它允许治疗结果的比较快速和清楚地表明，用标准数据表示另有遮蔽处理的细胞群内的显著异质性作图的强度数据以这种方式是很有用的。该反应的异质性是有趣在这个实验中，因为虽然一个统一的缺氧刺激是一个pplied到细胞中，显而易见的是，从同一个单层人口每一个细胞的反应不同，这种刺激。 图2D描绘了从我们的实验中，总体结果。从一个处理群中所有的细胞的荧光强度进行平均，然后绘制在该条形图。学生的t检验被用来计算每个治疗组从控制显著不同的统计概率。有趣的是，这些数据表明，有超过时U2OS细胞用缺氧处理的时间在全球RNA合成具有统计学显著增加（在时间大于1小时30分钟，* P &lt;0.01; ** p &lt;0.05和，*** P &lt;0.001和n&gt; 30）。这些数据表明，缺氧处理的时间较长，细胞论点集中，即使在如此恶劣的低氧环境下其对RNA合成的努力。放线菌素D处理完全烧蚀如预期般的荧光强度。<img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51420/51420fig1highres.jpg" src="/files/ftp_upload/51420/51420fig1.jpg" /> 图1:A的HIF系统响应的变化，细胞供氧。在这个简化的示意图缺氧稳定HIF二聚体通过防止HIF-α羟基（-OH）的脯氨酰羟化酶的酶（博士）及其以后的多泛素化被von-Hippel的林道蛋白（VHL）。缺氧诱导因子稳定在结果无数的基因，有助于可使用的Click-IT试验来检测B. RNA合成细胞缺氧细胞响应的激活;细胞治疗的Click反应检测RNA的合成可以通过荧光强度和光学显微镜进行量化后标有欧盟。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51420/51420fig1highres.jpg" target="_blank">请点击此处查看大图自愿减排量</a>离子这个数字。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51420/51420fig2highres.jpg" src="/files/ftp_upload/51420/51420fig2.jpg" /> 图2:从OMERO A.截图显示典型的微观输出下列RNA标记。这种显微镜图像可视化和分析软件，可以使用其内置的宏观投资回报率迅速提取物对单个细胞水平强度数据。从这个命令的输出的屏幕截图所示B.全球RNA的合成可以从单个细胞强度来推断，其范围可以在散点图可以看出C的这些汇总数据正式表示可以看出D. * P &lt;0.01; ** P &lt;0.05，*** P &lt;0.001和n&gt; 30。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51420/51420fig2highres.jpg" target="_blank">点击这里查看大图。</a> <tablë边境="0"cellpadding的="0"CELLSPACING ="0"&gt; <tbody><tr><td> 细胞培养试剂 </td></tr><tr><td>磷酸盐缓冲盐水（PBS）pH为7.2 - 7.6 </td></tr><tr><td> Dulbecco改良的Eagle培养基（DMEM）中</td></tr><tr><td>胎牛血清（FCS）的过滤除菌</td></tr><tr><td>青霉素/链霉素</td></tr><tr><td> L-谷氨酰胺</td></tr><tr><td> 0.05％胰蛋白酶-EDTA（1％） </td></tr><tr><td> 70％乙醇</td></tr><tr><td>放线菌素D（1微克/微升股票） </td></tr><tr><td> 细胞培养设备 </td></tr><tr><td>无菌巴斯德移液器</td></tr><tr><td>适合用于细胞培养的保养和治疗细胞培养处理过的塑料制品的一系列</td></tr><tr><td>置水浴在37℃ </td></tr><tr><td>空气层流罩</td></tr><tr><td>孵化器设置在37℃，5％CO 2的</td></tr><tr><td>缺氧工作站设置在37℃，5％CO2，1％O2 </td></tr><tr><td>盖玻片</td></tr><tr><td>精密镊子</td></tr><tr><td>血球</td></tr></tbody></table> 表1所需的U2OS细胞的培养试剂和设备。 <table border="0" cellpadding="0" cellspacing="0" width="514"><tbody><tr><td rowspan="2"> 反应元件 </td><td colspan="6"> 盖玻片数 </td></tr><tr><td> 1 </td><td> 2 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 10 </td></tr><tr><td>按一下，它的RNA反应缓冲液（成分C） </td><td> 428微升</td><td> 856微升</td><td> 1.7毫升</td><td> 2.1毫升</td><td> 2.58毫升</td><td> 4.3毫升</td></tr><tr><td> CuSO 4的（D成分） </td><td> 20微升</td><td> 40微升</td><td> 80微升</td><td> 100微升</td><td> 120微升</td><td> 200微升</td></tr><tr><td>的Alexa Fluor叠氮化物（制备步骤2.3） </td><td> 1.8微升</td><td> 3.7微升</td><td> 7.4微升</td><td> 9.3微升</td><td> 11.28微升</td><td> 18.8微升</td></tr><tr><td>点击 - 它反应缓冲液添加剂（准备步骤2.3） </td><td> 50微升</td><td> 100微升</td><td> 200微升</td><td> 250微升</td><td> 300微升</td><td> 500微升</td></tr><tr><td>概约总成交量</td><td> 500微升</td><td> 1毫升</td><td> 2毫升</td><td> 2.5毫升</td><td> 1.5毫升</td><td> 5毫升</td></tr></tbody></table> 表2的RNA成像套件反应鸡尾酒:参考表根据盖玻片被污染的数量，详细预混试剂进行RNA成像试剂盒的反应率。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 荧光 </td><td> 激励（纳米） </td><td> 发射（纳米） </td></tr><tr><td>的Alexa Fluor 488 </td><td> 495 </td><td> 519 </td></tr><tr><td>赫斯特33342，与DNA结合</td><td> 350 </td><td> 461 </td></tr></tbody></table> 表3荧光/激发发射峰:对的Alexa Fluor染料和赫斯特33342与DNA结合的近似荧光/激发发射最大。

Watch this Scientific Journal Video about Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia at JoVE.com

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

我们描述了全球性RNA合成缺氧使用成像分析技术。以前没有缺氧条件下进行的RNA点击化学标记，并允许全球的RNA变化的可视化在单细胞水平。这种方法将与现有的平均值RNA技术，允许的细胞 - 细胞间的变化在全球RNA合成的直接可视化。

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

analysis-global-rna-synthesis-at-single-cell-level-following

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

12.3K 次观看.  University of Dundee, UK. 我们描述了全球性RNA合成缺氧使用成像分析技术。以前没有缺氧条件下进行的RNA点击化学标记，并允许全球的RNA变化的可视化在单细胞水平。这种方法将与现有的平均值RNA技术，允许的细胞 - 细胞间的变化在全球RNA合成的直接可视化。 

视频: Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

病毒融合动力学测量方法在单粒子能级

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

科研

生物学

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

在大气压力质谱直接分析单细胞

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

单细胞基因转录的RNA荧光分析<em在原位</em&gt;杂交（FISH）

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

在整个细胞群在单细胞水平上研究细胞周期蛋白B的蛋白水解

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

在EML造血前体细胞调节自我更新和分化的关键因素，通过RNA测序分析鉴定

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Here we employ FISH methodology to track LINE-1 retrotransposition at the single nuclei level in chromosome spreads of HepG2 cell lines stably expressing synthetic LINE-1.

在单核级LINE-1逆转录转座子分析

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

CUBIC协议可视化在全山皮肤配方单细胞分辨率蛋白表达

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

斑马鱼胰岛单细胞分辨率钙成像对β细胞功能的分析

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified &#955; DNA at the Single Molecule Level

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. In single molecule assays for studying DNA-protein interactions, there are two important factors for consideration: the DNA substrate with enough length for easy observation and labeling a protein with a suitable fluorescent probe. 48.5 kb &#955; DNA is a good candidate for the DNA substrate. Quantum dots (Qdots), as a class of fluorescent probes, allow long-time observation (minutes to hours) and high-quality image acquisition. In this paper, we present a protocol to study DNA-protein interactions at the single-molecule level, which includes preparing a site-specifically modified &#955; DNA and labeling a target protein with streptavidin-coated Qdots. For a proof of concept, we choose ORC (origin recognition complex) in budding yeast as a protein of interest and visualize its interaction with an ARS (autonomously replicating sequence) using TIRFM. Compared with other fluorescent probes, Qdots have obvious advantages in single molecule studies due to its high stability against photobleaching, but it should be noted that this property limits its application in quantitative assays.

Here, we present a protocol to study DNA-protein interactions by total internal reflection fluorescence microscopy (TIRFM) using a site-specifically modified &#955; DNA substrate and a Quantum-dot labeled protein.

Qdot 标记蛋白与现场特异修饰的λ DNA 在单分子水平上的相互作用可视化

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. ...