This work was supported by the following grants: AHA 16SDG31170008 to X.R. and NIH P20 GM109098, P01 AG027956, and U54 GM104942 to J.W.S.

注:该协议的方案示于图1，其中包括的步骤的时间表。 1.文化和CVE细胞通道<ol><li>培养CVE细胞（bEnd.3细胞）在完全生长培养基（高葡萄糖的Dulbecco改良的Eagle氏培养基，DMEM）中补充有10％胎牛血清（FBS）和1％青霉素/链霉素的T75厘米2组织培养烧瓶中。在一个5％CO 2培养箱生长于37℃。 </li><li>卸下并丢弃细胞培养基。冲洗用0.25％胰蛋白酶0.03％的EDTA溶液的细胞层，然后添加1 - 2毫升胰蛋白酶-EDTA溶液到烧瓶中，并保持该烧瓶在37℃，3 - 5分钟。观察的倒置显微镜下细胞直至细胞层被分离。加入10 mL的完全生长培养基中，并轻轻吹打吸出细胞。 </li><li>滗流体和细胞进入有15毫升离心管中并旋转，在700×g离心5分钟，除去细胞碎片。 </l我&gt; <li>丢弃从离心管的上清液。加入10 mL的完全生长培养基中，并重新悬浮细胞沉淀。旋转细胞在700×g离心5分钟。 </li><li>丢弃从离心管的上清液。重悬在完全生长培养基将细胞沉淀在2.0×10 5个细胞/ mL的浓度（通过使用血细胞计数器计数测定;可以通过台盼蓝染色排除死细胞）。 注:新鲜解冻细胞对于至少3个通道用于实验亚培养。 </li></ol>在细胞培养板2.种子和细胞治疗<ol><li>上的96孔细胞外磁通细胞培养板的种子细胞:80微升/孔。放置在37℃的细胞培养板在5％CO 2的培养箱中。注:每孔16×10 3个细胞将达到60 - 24小时内80％汇合。 </li><li>如果在细胞被暴露于化学，添加处理一旦将细胞附着在板的底部（8 - 24小时后看到鼎）。 注:对于转染实验中，不完全生长培养基中添加抗生素。 </li></ol> 3.线粒体功能的评价使用生物分析仪<ol><li>前测定的开始，通过加入外磁通校准溶液的150微升到板，并在37℃下无CO 2的孵育O / N水合的一个细胞外磁通传感器盒与校准。 </li><li>使用洗板站，细胞培养基改变成pH为7.0的细胞外磁通测定培养基。孵育在37℃的细胞无CO 2的30 - 60分钟。 </li><li>准备8μM寡，4.5微米FCCP，10μM鱼藤酮，并在10 DMEMμM抗霉素A的工作方案。装载25μL储备液成盒注射端口:端口A，寡;端口B，FCCP;端口C，鱼藤酮和抗霉素A与细胞外通量分析网上平台。如表1中所述实施分析方案。 </li><li>加载水合盒插入生物分析仪，并通过单击"开始"按钮进行校准。校准完成后，取出硒鼓底板，然后通过单击"卸载磁带"的提示加载单元板。继续试验直至所有测量的结束。 </li><li>打开数据文件并获得生物分析仪的速度值。将数据导出到电子表格文件。 </li></ol> 4.数据分析注:数据分析的计算如下制定。作为OCR 图2中呈现的值是从生物分析仪获得的。 <ol><li>为了计算基础呼吸 ，减去非线粒体呼吸（测量10 - 12）从基础值（测量3）。 注:基础呼吸=基础 - 非线粒体呼吸=率③ - 费率⑩⑫〜。 </li><li>为了计算ATP生产图片上，减去的ATP联呼吸（测量4 - 6）从基值（测定3）。 注:ATP生产=基础 - ATP挂钩呼吸=率③ - ④率⑥〜。 </li><li>要计算最大呼吸 ，减去非线粒体呼吸（测量10 - 12）最大呼吸（测量7 - 9）。 注:最大呼吸=最大呼吸 - 非线粒体呼吸=率⑦〜⑨ - 费率⑩⑫〜。 </li><li>要计算余力 ，减去最大呼吸基础值（测量3）（测量7 - 9）。 注:备用容量=最大呼吸 - 基底=率⑦〜⑨ - 率③。 </li><li>计算质子泄漏 ，减去非线粒体呼吸（测定10 - 12）从ATP联呼吸（测定4）。 注:质子漏= ATP挂钩呼吸 - 非线粒体升呼吸率=④ - 费率⑩⑫〜。 </li></ol>

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The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Open Access fees for this article were provided by Agilent Technologies.

该协议代表了脑血管内皮细胞的生物能量表型的评估的方法。它用作用于内皮细胞线粒体评价基本分析，它是最佳的设计，调查刺激可能影响在CVE细胞线粒体信号传导途径的机制的实验。它也提供了用于测试可能的治疗剂为BBB-破坏相关疾病的方法。 该议定书中的关键步骤胞磁通bioanalyzers必须测量的OCR实时能力。在该测定中，确定适当的细胞密度是至关重要的。如果细胞密度低于每孔8×10 3个细胞，基底OCR太低待分析（ 图2和图3）;如果细胞密度高于每孔32×10 3个细胞，该细胞不响应寡或FCCP treatmeNTS（ 图2和图3）。在我们的实验模型，该细胞系的最佳细胞密度为每孔，这表明复制的结果和敏感性刺激7和治疗10,14 16×10 3细胞。如果使用24孔生物分析仪，细胞密度的新滴定将需要用于测定。 据记载，的CVE具有大的体积线粒体与其它内皮细胞或组织类型15,16相比，表明更大的能源生产和更少的细胞，用于测量生物能量代谢的需要。我们已经测试了几种类型的脑内皮细胞，如初级和永生化的鼠脑的内皮细胞7和永生化人脑内皮细胞（未公布的数据）。要求这些细胞相似的细胞密度40内达到可接受的OCR（基础呼吸OCR - 160皮摩尔/分钟96孔板和50 - 400皮摩尔/ 24孔板分钟）时，进行的生物能学测定法。 Kaczara 等报道测量生物能量代谢中的人脐静脉内皮细胞（HUVEC），它使用了更高的细胞密度17。 我们的团队没有评估来自其他组织或器官血管细胞。从理论上讲，该生物分析仪可能被在任何细胞类型使用，但它需要优化细胞密度，细胞培养基，和培养条件（某些特定的细胞可能需要涂层板生长良好）测定。它要求该实验完成，以确保OCR率在可接受的范围之内。如果OCR在从其它器官的内皮细胞比从大脑的CVE下，增加细胞密度可以帮助到可接受的OCR的范围内得到。另外，如果电池不使用氧化磷酸化作为其主要能源，生物分析仪不会是一个最优升检测。 生物分析仪允许电子传递链的连续中断，基于在施加试剂的顺序。首先，寡被应用，其抑制线粒体复合物V（ATP合酶）。第二，FCCP，电子解偶联剂被施加，这导致质子梯度的干扰。最后，鱼藤酮和抗霉素A，其抑制线粒体复合物I和III分别被施加到导致电子流的总抑制。药物暴露的顺序是必要的，因为该药物阻断的特定电子传输链反应，和顺序的变化可以被测量以反映线粒体功能。 修改和故障排除以评估线粒体响应在CVE细胞对刺激，则建议的刺激被（见时间轴施加到细胞后的细胞粘附到细胞培养板底部在图1中示出;它通常需要至少6小时）。由处理获得可复制的结果，以保持细胞密度一致是非常重要的。如果预处理之前将细胞接种设计的，对于每个前处理细胞密度应仔细测量，但不包括用于播种的死细胞。如果转染是包含在实验设计，请参照制造商提供的转染方案。在图4给出的数据证明中，miR-34a的转染到使用脂质体转染试剂盒，这要求无抗生素的细胞培养基和丝裂应力测试用miR-34a的表达，以评估在代谢功能的变化CVE细胞。质粒的剂量也可以被优化，如先前发表13。可以对协议进行另一个变化是改变试剂的浓度。寡，FCCP，和/或鱼藤酮和抗霉素A的滴定曲线可以完井特德。 此外，如果各种药物该测定被用于高通量分析，建议包含一个平行试验，以测量细胞生存力和细胞增殖，这是在我们以前的出版物7,13-说明。 OCR的值由细胞数目（ 图2和3）显著的影响，并在细胞增殖和活力测定法中获益的数据的正常化。然而，这些试验的完成是在每个单独的实验室的判断。 该技术的局限性这个协议的主要限制是，我们仅仅在研究中使用的体外细胞培养物模型。目前，没有可用的体外模型或动物模型可针对内皮细胞的线粒体功能。预期新的模型生物能量函数的体内评估待开发和<eM&gt;体外在未来的研究。 另一个限制是继生物能措施的障碍评估的扩展。因为，首先，将生物能量测量完成后，细胞存活率减弱的电子传递链的完整的中断不能进行的实验;第二，需要特定的细胞培养板和插入检测生物能量值，不适合障碍评估插入。因此，没有能生物能学分析后可以完成很多功能分析。然而，可以预期的特殊装置，可开发来执行前的生物能测定功能测定。 关于到现有/替代方法的技术意义先前的技术来评价线粒体功能必要线粒体的分离从细胞18。该网元使用生物分析仪W¯¯技术允许完整细胞线粒体活性，它保留超过评估分离线粒体细胞环境的测量。 掌握这一技术后，未来的应用方向或该协议被设计和用于bEnd.3细胞系开发的，但它也与初级脑血管内皮（pCVE）细胞7或其他内皮兼容，我们已经在我们以前使用pCVE电池7出版物表明了这一点。当使用其他类型的内皮细胞的，可能需要在培养板的涂布和使用的生长因子。然而，滴定试验被推荐用于其它细胞类型为好。这个协议提供了在CVE细胞的生物能学的评价将要通过通常的方法，它可以进一步施加到机构的研究或以这种方式治疗反应。

人们普遍认识到，通过脑血管的内皮（CVE）细胞所形成的血 - 脑屏障（BBB）具有极为重要的血管生物学非常不同和独特的功能。这些内皮细胞使用线粒体生成最三磷酸腺苷的细胞供应（ATP）的化学能量源。除了用于CVE细胞中提供的ATP，线粒体调节在血管内皮细胞，如细胞信号1-4的各种细胞过程，细胞凋亡5，和细胞周期控制和细胞生长6。在CVE细胞线粒体生物能量功能的失调可能影响线粒体生物合成，从而导致受损血管内皮活性，并加剧脑血管疾病和神经变性疾病， 如中风7,8和阿尔茨海默氏病（AD）9。我们已经表明，暴露于lipopolysac侮辱CVE细胞charide（LPS）损害的CVE细胞线粒体功能和恶化中风成果7。叔丁基氢醌（TBHQ）通过在CVE电池10的氧化磷酸化的干扰增加中风的死亡率。因此，生物能量功能的CVE细胞不仅导频用于中枢神经系统的一系列机制相关的研究（CNS）的疾病的定量模型，而且还提供了在治疗中血管生物学靶向线粒体功能的药物筛选的一个突破。 分离线粒体已被用来测量生物能的功能。我们7和其他11个通过使用外通量生物分析报告完整CVE细胞生物能量的细胞的评估。该分析仪提供了内源性细胞生物能量评估的优势。这个敏感和一致的测定测量CVE细胞的线粒体代谢和可用于评估由STI生物能障碍木里，调查线粒体信号通路的影响在CVE细胞线粒体功能的机制，和屏幕药物或治疗。耗氧率（OCR）是通过使用药理分析方法相结合，使用四个线粒体干扰生物分析仪记录:寡，羰基氰化物-4（三氟甲氧基）苯腙（FCCP），鱼藤酮，抗霉素和A.在该协议中，我们详细的优化方法，使该测量和CVE细胞线粒体机能参数，包括基底线粒体呼吸，ATP产生，质子泄漏，最大呼吸，和备用呼吸容量的计算，在一个单一的检测。

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		<tr height="26">
			<td height="26" style="height:27px;width:377px;">bEnd.3 cell line</td>
			<td style="width:147px;">ATCC</td>
			<td style="width:119px;">CRL-2299</td>
			<td style="width:197px;">25-30 passages</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td style="width:147px;">ATCC</td>
			<td style="width:119px;">30-2002</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Fetal bovine serum</td>
			<td style="width:147px;">Atlanta Biologicals</td>
			<td style="width:119px;">S12450</td>
			<td>10% in final</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Penicillin/Steptomycin</td>
			<td style="width:147px;">Hyclone</td>
			<td style="width:119px;">SV30010</td>
			<td>1&#215;100 stocking</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">0.25% trypsin 0.03% EDTA solution</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">25-053-CI</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Sodium pyruvate</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">25-000-CI</td>
			<td>1.0 &#181;M in final</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Glucose</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:119px;">CAS 50-99-7</td>
			<td>25 mM in final</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Oligomycin</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:119px;">O4876</td>
			<td>1.0 &#181;M in final</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Carbonilcyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:119px;">C2920</td>
			<td>0.5 &#181;M in final</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Rotenone</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:119px;">R8875</td>
			<td>1.0 &#181;M in final</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Antimycin A</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:119px;">CAS 1397-94-0</td>
			<td>1.0 &#181;M in final</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Plate wash station</td>
			<td style="width:147px;">Seahorse Bioscience</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Extracellular flux bioanayzer&#160;&#160;</td>
			<td style="width:147px;">Seahorse Bioscience</td>
			<td style="width:119px;">XFe96</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Extracellular flux cell culture plate</td>
			<td style="width:147px;">Seahorse Bioscience</td>
			<td style="width:119px;">102416-100</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Extracellular flux sensor cartridge</td>
			<td style="width:147px;">Seahorse Bioscience</td>
			<td style="width:119px;">102416-100</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Extracellular flux calibrant solution</td>
			<td style="width:147px;">Seahorse Bioscience</td>
			<td style="width:119px;">100840-000</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:377px;">Extracellular flux assay medium</td>
			<td style="width:147px;">Seahorse Bioscience</td>
			<td style="width:119px;">102365-100</td>
			<td>PH buffered prior to assay</td>
		</tr>
	</tbody>
</table>

evaluation of bioenergetic function in cerebral vascular endothelial cells

血 - 脑屏障（BBB）的完整性是关键的，以防止脑损伤。脑血管内皮（CVE）细胞是包含血脑屏障的细胞类型中的一种;这些细胞具有非常高的能量需求，这需要最佳线粒体功能。在疾病或损伤的情况下，在这些细胞中的线粒体功能可以被改变，从而导致疾病或血脑屏障的开口。在这个手稿中，我们将介绍使用的整体，完整的细胞和生物分析仪测量CVE细胞线粒体功能的方法。一个三刀应力测定是用来挑战已经被扰动，物理或化学的细胞，并评估其生物能量的功能。此外，这种方法还提供了筛选具有对线粒体功能的直接影响新的治疗剂的有用方法。我们优化所需的细胞密度，以产生氧消耗速率，其允许多种线粒体段的计算平方米，包括ATP的产生，最大的呼吸和备用容量。我们还通过证明引入微小RNA中，miR-34a的，导致线粒体活性显和检测到的降低表明该测定的灵敏度。而在本文中示出的数据是针对bEnd.3细胞系的优化，我们还进行了优化初级CVE细胞协议，进一步表明在临床前和临床模型的效用。

评估响应于氧化应激CVE细胞的生物能量函数，我们选择了鼠脑微血管内皮细胞系bEnd.3，其显示相同的屏障特性如原发性脑微血管内皮细胞12。鉴于动力学和响应的相对强度是在各种细胞类型中改变，第一系列的实验中，设计通过识别在测定代谢分析使用bEnd.3细胞的最佳数量，以获得可测的OCR的水平，显示在图2.继评估，数据被量化，并在图3中。基础呼吸，最大呼吸呈现，和备用呼吸容量显示出与细胞密度的比例响应。然而，当选择了每孔64×10 3个细胞的ATP产量下降，这表明在汇合细胞培养物是不适合于该实验。最优细胞密度发生8之间-每孔的细胞的32×10 3个的基础上，响应于线粒体干扰。 对于随后的实验，每孔16×10 3个细胞的接种密度被用来允许在OCR变化的最佳检测。用每孔16×10 3个细胞，我们观察到在OCR所期望的响应，并表明微RNA的miR-34a的减少CVE细胞线粒体功能（ 图4）。我们以前曾报道了miR-34a的在这些细胞中13减少氧化磷酸化。重复实验还表明，最大呼吸和备用容量由在的CVE的miR-34a的表达在24小时后转染显著下降，即使基础呼吸，ATP产生，和质子漏无显著变化（ 图4） 。这些数据证明生物分析仪的灵敏度来检测的CVE中线粒体功能的变化。 <img alt="图1" src="/files/ftp_upload/54847/54847fig1.jpg" /> 图1.战略规划实验。时间线为单元，板和墨盒准备指示。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54847/54847fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54847/54847fig2.jpg" /> 在CVE细胞不同的细胞密度测量图2. CVE生物能细胞功能的各种细胞密度的代表性原始数据。耗氧率。数据代表平均值±标准差（n = 5）; OCR:耗氧率; FCCP:羰基氰化物-4-（三氟甲氧基）苯腙;<html>腐/防答:鱼藤酮和抗霉素A.①，②，③和基础注明呼吸; ④，⑤，⑥和ATP注明链接呼吸; ⑦，⑧，⑨和最高指示呼吸; ⑩，⑪，⑫和指示非线粒体呼吸。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54847/54847fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54847/54847fig3.jpg" /> 图3.在用各种细胞密度CVE细胞生物能功能的代表结果。基底呼吸，ATP产生，最大呼吸，和备用容量是从由生物能产生的原始数据计算出的图使用公式2功能测定在第4所示。数据代表平均值±标准差（n = 5）; OCR:耗氧率。<html> F ="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54847/54847fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54847/54847fig4.jpg" /> 图4:受miR-34a的生物能降低功能代表性的成果 （A）以下24小时后转染的miR-34a的表达线粒体功能的原始数据。 （B）的基础呼吸，ATP产生，最大呼吸，和备用容量是从由生物能在图4A功能测定法产生的原始数据计算;该参数是由在第4节的miR-34a的表达所指示的公式来计算减少了在24小时后转染CVE细胞线粒体功能。数据代表平均值±标准差（n = 5）; OCR:耗氧率。 ****，P &lt;0.0001。文件/ ftp_upload / 54847 / 54847fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="表格1" src="/files/ftp_upload/54847/54847table1.jpg" /> 表1仪器运行协议。

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Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

内皮细胞线粒体是维持血脑屏障完整性的关键。我们引入一个协议来测量脑血管内皮细胞生物能量的功能。

在脑血管内皮细胞生物能量功能的评价

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

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9.2K 次观看.  West Virginia University. 内皮细胞线粒体是维持血脑屏障完整性的关键。我们引入一个协议来测量脑血管内皮细胞生物能量的功能。 

视频: Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

突触小泡池，利用苯乙烯染料的光转化的研究

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

科研

神经科学

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

原代鼠脑微血管内皮细胞的分离

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

利用双光子显微镜的艾滋病毒引起的神经炎症的小鼠模型脑血管结构的量化

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

影像学检查HT能纤维在小鼠脊髓使用CLARITY / CUBIC技术

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

脑血流动力学和的光散射特性的同步评估<em&gt;体内</em&gt;大鼠脑使用多光谱漫反射成像

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund&#39;s adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter.

由亲炎细胞因子的内脑注射诱导的广度脑皮质脱叶的鼠模型

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

幼虫斑马鱼外系头发细胞的活体钙成像

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

原代小鼠骨骼肌微血管内皮细胞的分离

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...