We thank J. Schmidt from the University of Freiburg for constructing the device. We thank P. Nielsen and D. Medgyesi for their support and critical reading of this manuscript. This study was supported by the Deutsche Forschungsgemeinschaft through SFB746 and by the Excellence Initiative of the German Research Foundation (GSC-4, Spemann Graduate School).

1.设计和建造设备<ol><li> 试点实验 注:为特定的光遗传学工具和细胞类型所需要的光照强度可能会有所不同显著。与原型试点实验是估计所需的最低光照强度是有用的。用于以下实验的光遗传学工具是Dronpa - 接头 - Dronpa融合构建。从商业上获得Dronpa序列（参见材料清单）。长接头克隆两个Dronpa序列之间，以允许表达构建体，以形成分子内（分子间）二聚体。下面描述的步骤可以适于许多其它光遗传学工具或光控制物质。 <ol><li>转导拉莫斯B淋巴细胞与使用含逆转录病毒的上清液从包装细胞按照制造商的转导的指令的Dronpa - 接头 - Dronpa含有构建体。采收和计数细胞根据预先viously发布的协议13。 </li><li>重悬的细胞以浓度1×10 6 / mL的培养基（RPMI 1640,10％热灭活的FCS，2mM的L-谷氨酰胺，100U / mL青霉素，100U / mL链霉素，和50μM2-巯基乙醇）并把它们转移到一个玻璃试管FACS。 </li><li>插入管插入流式细胞仪和测量Dronpa（GFP通道）14,15的荧光强度。 </li><li>穿所有进一步的措施防护眼镜，以保护眼睛免受可能会损坏波长。 </li><li>手动放置在管的外侧上的500nm的LED光，并继续测量Dronpa的荧光强度。 </li><li>增加LED灯的数量和/或强度，直到Dronpa荧光强度降低。 </li><li>手动放置在管的外侧上的400nm的LED光，并继续测量Dronpa的荧光强度。 </li><li>增加LED灯的数量和/或强度，直到Dronpa FLUOrescence强度增大。 </li><li>至少使用尽可能多的LED灯作为预测是必要的步骤1.1.5和1.1.7的建设光控设备。 </li></ol></li><li> 建筑设备 注:该设备是由一个专业的机械加工车间，Arbeitsgruppe技术公司从弗莱堡大学建成。大部分地区都是定制的，而不是商业化。各部分的精确的尺寸在图1和2中描绘。为了澄清本装置的组装，补充视频1被提供并最本质的步骤如下所述。 <ol><li>磨片A从聚氯乙烯（PVC），与图1A中所示的精确的测量。 </li><li>将LED灯成片A的钻孔和密封用PVC胶水每孔防止水的渗漏。 </li><li>连接LED的多通道转换器（每个波长应该占据一个独立的陈荫罴EL）与0-29毫安的可调输出功率。 </li><li>附加的外鞘（B）和3螺钉片A如所描绘，以保护LED不受物理损坏。 </li><li>为了能够将设备连接到一个水泵，胶水在管夹（C）的成片A的钻孔（ 图1a所描述的，交叉部Y为9mm 61.5 MM）使用PVC胶水。 </li><li>削减58.5毫米长的有机玻璃管（D1）。 </li><li>从有机玻璃制造件D2和D3， 如图2中所描绘。 </li><li>胶块D3至D1的底部有机玻璃胶水。 </li><li>附加橡胶O型圈到一块D2，它胶水D1的顶部，有机玻璃胶水。 注:作品D1，D2和D3一起形成片D. </li><li>插入件d成片带3个螺丝。 </li><li>测试之前，将电源变压器渗漏水的设备。 </li></ol></li></ol> 2.测量的光遗传学工具的动力学研究<ol&gt; <li> 准备细胞 <ol><li>培养拉莫斯B细胞在RPMI培养基（RPMI 1640,10％热灭活的FCS，2mM的L-谷氨酰胺，100U / mL青霉素，100U / mL链霉素，和50μM2-巯基乙醇）中的3-密度10×10 6个细胞/ mL，在37℃和5％的CO 2。 </li><li>收获拉莫斯B细胞通过离心在350 XG和4℃5分钟。 </li><li>算使用Neubauer计数室按照制造商的指示细胞，重悬1-5×10 6个细胞在600微升培养基。 </li><li>转移细胞的玻璃FACS管，把它们放在冰和保护他们免受光直到测量。 </li></ol></li><li> 制备流式细胞仪和设备。 <ol><li>将设备连接到一个加热的水泵和调节温度至37℃。 </li><li>当使用玻璃FACS管，更换流通的黑色O型圈仪用橡胶O型圈和额外使用硅GR缓解。 </li></ol></li><li> 测量 <ol><li>小心地将玻璃FACS管进入设备，并将其连接到流式细胞仪（ 图3）流动。 </li><li>开始测量，并记录Dronpa荧光强度（GFP通道）。 </li><li>分别照亮为400nm或500nm的光的细胞样品，并记录Dronpa荧光强度（GFP通道）。 </li></ol></li><li> 数据分析 <ol><li>分析用合适的流式细胞仪软件的实验数据。对侧向散射和门活细胞情节前向散射。绘制随着时间的推移Dronpa荧光强度。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

LED的热流量是一个创新的设备，研究光遗传学工具在流式细胞仪。 到目前为止，光遗传学样品已经只能用显微镜激光或闪光装置11,12照明。根据不同的手电筒与样品的角度和距离，在光照的量实质性变性实验之间的预期。此外，存在于手电筒一个人可以在实验操作的数目的限制。这限制了实验剧目和重现性。这些限制我们的装置，该装置可用于在活细胞中表征实时光控动力学的发展过程中进行了讨论。据我们所知，没有类似的设备存在。 在当前的配置，多达30个LED可以被做成热流室。因此，根据所要求的光强度对每个波长，单个设备可以是我们编为各种各样的光遗传学工具。该光控建立协议可以被用于功能读数， 如钙通量测量优化。我们的设备可以适用于其他光控制物质，如笼化合物或光活化荧光团。 根据科学问题和使用试样，个性化协议可以迅速建立。使用玻璃试管代替聚苯乙烯或聚丙烯管的建议在400-500纳米之间的波长，以限制光诱导的细胞毒性。所有测试的细胞系存活照度（360纳米，400纳米或500纳米），用于在玻璃管长达一小时。我们试图通过执行转移实验来研究细胞死亡的塑料管照明中的原因。我们照亮在PBS或RPMI细胞用不同波长的光，然后转移上清至非照射细胞来测量细胞死亡。所收集的无上清液在受体细胞引起显著的细胞死亡（数据未显示）。另外，照射的PBS在聚苯乙烯或聚丙烯管中的温度几乎是相同的在玻璃管中的温度。因此，我们只能推测细胞死亡的原因。照亮塑料可以释放出波长是毒性细胞的不稳定物质或辐射。 介质的用于每个实验的选择是重要的。缓冲能力必须考虑和不同的试剂，如pH指示剂，吸收不同波长的程度不同。此外，比较自由FCS的时到含有FCS的培养基的细胞存活显著不同，例如，。 光滴定的目标是使用必要的最大光控光的最小量。对于大多数实验中，这是有利的最大化光电开关的速度，从而最大程度地光强。但是，根据不同的waveleNGTH和细胞类型，光可以具有在所述信令行为，这就是为什么过度照明应避免直接的影响。 有可能对我们的装置的光的时间表编程和将它连接到一台计算机为其他光装置普遍。然而，由于大多数流式细胞仪是由许多不同的实验室共同的工作场所时，最好保持装置小巧便携越好。此外，我们对这里所提出的两颜色设置设备的手工操作就是这么简单，那将编程仅略有提高实验过程。 总之，我们在这里提出了一个创新的设备，它结合了光遗传学工具的力量和流式细胞仪。这将大大简化在体内的表征和光遗传学工具的开发和拓展，实验剧目。

光遗传学工具已日益普及，部分是因为它们可用于破译信号通路1-4的布线。它们是基于可光活化的蛋白质当与光照射改变其构象和结合​​亲和力的能力。融合这些蛋白质信令元素允许单个玩家的复杂细胞内信号通路5-12内的特定调节。因此，信号传导途径可以以高时空分辨率进行研究。 大多数基于细胞的光遗传学研究利用基于显微镜的方法，在光的存在下培养相结合，随后通过生物化学分析11,12。与此相反，一个流式细胞singularizes细胞沿着毛细管并测量细胞大小，粒度和荧光强度。这种方法具有在显微镜或生物化学方法主要优点，包括分析thousan的能力家里单细胞分辨率的细胞在非常短的时间的DS。因此，理想的是光遗传学流式细胞仪结合。 据我们所知，有一个光遗传学流式细胞仪没有既定的协议。一个被广泛接受的过程是从手电筒设备反应管外手动点亮照明单元。然而，在流动手册照明仪要求光通过反应管，并用于活细胞成像，圆柱形，加热的水室中。这会导致大量的光散射和光的损失。此外，通过手动照明提供的光强度不实验（角度，距离， 等等 ）之间的可再现的和有一个实际的限制，以波长的数量在一项实验。 通过构造该LED热流量设备，我们能够克服这些限制。与此设备中，细胞可以在一个临时的特定波长照射erature控制在流式细胞仪的测量方式。这使得在与实验之间的精确和可重复的光量。 为了证明我们的体内装置的效用，我们在光控记录在拉莫斯B细胞Dronpa的荧光信号。拉莫斯B细胞是从人Burkitt淋巴瘤的。 Dronpa是存在作为单体，二聚体或四聚体的荧光蛋白质。在它的单体形式，它是无荧光的。照明用400纳米的光诱导二聚和四聚并呈现Dronpa蛋白荧光。这个过程可以通过用500nm的光照射可以相反。该Dronpa蛋白已被用于控制信号蛋白4,13的功能和位置。 在此，我们表示拉莫斯B细胞Dronpa连接子Dronpa蛋白研究Dronpa光控的在流式细胞仪。使用我们的装置中，我们能够有效地和重复性光控Dronpa同时记录实时荧光强度。这种方法比用人工照明照度当前的协议提供了巨大的优势和显著扩大了光遗传学工具和笼化合物的实验剧目。使用我们的设备将显著简化并加速的新颖光遗传学工具的发现和开发。

<table><tbody><tr><td>Glass FACS tube</td><td>Thermo Fisher Scientific; Waltham, USA</td><td>14-961-26</td><td>Borosilicate glass tubes 12x75 mm</td></tr><tr><td>Flow Cytometer</td><td>BD Bioscienceg; Heidelberg, Germany</td><td>Fortessa II</td><td>Special Order</td></tr><tr><td>Dronpa: pcDNA3-mNeptune2-N</td><td>Addgene; Cambridge, USA</td><td>41645</td><td></td></tr><tr><td>PolyJet</td><td>SignaGen, Rockville, USA</td><td>SL100688</td><td></td></tr><tr><td>LED 505 nm</td><td>Avago Technologies; Boeblingen, Germany</td><td>HLMP-CE34-Y1CDD</td><td></td></tr><tr><td>LED 400 nm</td><td>Avago Technologies; Boeblingen, Germany</td><td>UV5TZ-400-15</td><td></td></tr><tr><td>Plexiglas tube 15 mm</td><td>Maertin; Freiburg, Germany</td><td>76999</td><td></td></tr><tr><td>Plexiglas 3 mm</td><td>Maertin; Freiburg, Germany</td><td>692230</td><td></td></tr><tr><td>Plexiglas 2.5 mm</td><td>Maertin; Freiburg, Germany</td><td>692225</td><td></td></tr><tr><td>Plexiglas 1.5 mm</td><td>Maertin; Freiburg, Germany</td><td>692215</td><td></td></tr><tr><td>PVC tile 5 mm</td><td>Maertin; Freiburg, Germany</td><td>690020.005</td><td></td></tr><tr><td>PVC tile 6 mm</td><td>Maertin; Freiburg, Germany</td><td>690020.006</td><td></td></tr><tr><td>PVC block 50 mm</td><td>Maertin; Freiburg, Germany</td><td>690020.050</td><td></td></tr><tr><td>RPMI</td><td>Invitrogen, Life Technologies; Darmstadt, Germany</td><td>61870-010</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>EMD; Germany</td><td>805740</td><td></td></tr><tr><td>FCS</td><td>PAN Biotech; Aidenbach, Germany</td><td>P30-3302</td><td></td></tr><tr><td>Penicillin/Strptomycin (10,000 U/mL)</td><td>Invitrogen, Life Technologies; Darmstadt, Germany</td><td>15140-122</td><td></td></tr><tr><td>Acrifix plexiglas glue</td><td>Evonic industries, Essen, Germany</td><td>1R0192</td><td></td></tr><tr><td>Tangit PVC-U glue</td><td>Henkel, D&amp;uuml;sseldorf, Germany</td><td></td><td></td></tr></tbody></table>

led thermo flow &#8212; combining optogenetics with flow cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

使用LED热流量用流式细胞仪该装置的功能的核心是一个圆筒形室，其中LED灯被布置在向内指向循环方式。这种腔室可连接到水供应和泵，其允许控制LED的温度，以及所述细胞样本。 LED被连接到变压器，从而为各波长的光强度可被单独控制。该装置的中心容纳一个标准大小的FACS管，其可以连接到最标准的流式细胞仪（ 图4和5）。要显示在我们的设备不导致细胞的不希望的加热而照明，我们测量照亮的PBS随时间的温度（ 图6）三种不同的波长。照明用360纳米，400纳米或500纳米导致只有一个边际温度上升。总体上，这里提出的装置允许用于温度控制的细胞样品的再生照明，以流式细胞仪进行测量。 Dronpa光控的编码Dronpa -接头- Dronpa 13,16融合构建的表达载体被克隆和转导到拉莫斯B细胞。 如图7中所描绘的，目的是控制这种融合蛋白的构象具有特定波长的光。使用我们的装置中，胞质Dronpa -接头- Dronpa融合蛋白photoswitched多次（ 图8，左）。切换到暗状态时慢，而切换到明亮状态发生几乎瞬间。在黑暗Dronpa的自发荧光恢复显示，光控效率，以明亮的形式需要特定的照明和只能慢慢自发地发生（ 图8，右）。 我们进行的光控性能的动力学分析（ 图9）。用于切换动力学的斜率计算出照明的初始4分钟。对于自发恢复斜率计算在黑暗的初始4分钟。 动力学分析表明，该光控Dronpa速度整体非常重现性。与此相反，在黑暗中Dronpa荧光的自然恢复是非常低效的。即使在黑暗中再温育后，只有约10％的Dronpa荧光的恢复。这表明的确在我们的装置中的照明诱导光控。 因此，使用所描述的方法，我们可以生成实时在流式细胞仪光控数据，从而可以被翻译成的许多光遗传学工具的动力学评价。 <img alt="图1" src="/files/ftp_upload/54707/54707fig1.jpg" /> 图1: 外层的建设计划。所有描绘片定制从聚氯乙烯（PVC）制成。 片A:圆柱PVC片与钻孔插入LED和管夹。 件B:外鞘，以保护LED不受物理损坏。 件C:该管夹连接用橡胶管内腔以允许水冷却。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54707/54707fig1large.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/54707/54707fig2.jpg" /> 图2:内层的建设计划。<html>所有描绘片由有机玻璃制成。 片D:此片是由三片D1，D2和D3与构成单元包围的FACS管。 件D1:圆柱形有机玻璃管。 件D2:内层的底部盖。 件D3:内层的盖子。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54707/54707fig2large.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/54707/54707fig3.jpg" /> 图3: 使用设备用流式细胞仪。 A1:流式细胞仪的样品入口; A2:连接到样品入口FACS管; A:封闭管FACS设备; C:样品入口; D:样品入口与设备。ove.com/files/ftp_upload/54707/54707fig3large.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/54707/54707fig4.jpg" /> 图4: 工作流的设备的示意图。 答 :将细胞在该装置照亮（左下），并同时吸收并在流式细胞仪分析（左上）。这允许，例如，一个光遗传学工具（右上）的动力学性质的分析。 A:该装置的横截面。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54707/54707fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54707/54707fig5.jpg" /> 图5:该装置的工艺流程图。一个或MOR<html>特定波长的E LED灯照亮了FACS管细胞样品。这些LED是由水，这是不断被加热浴循环交换，以保持细胞样品稳定的温度所包围。活细胞的实验中，将温度调节到37℃。在此温度控制照明，将细胞吸收到流式细胞仪进行分析。这种设置允许，例如，分析一个光遗传学工具的动力学性质。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54707/54707fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54707/54707fig6.jpg" /> 图6:随着时间的推移照明PBS的温度。 1毫升的PBS中的玻璃的FACS管中随着时间的推移与360纳米的光，400nm的光或500nm的光照射。上传/ 54707 / 54707fig6large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图7" src="/files/ftp_upload/54707/54707fig7.jpg" /> 图7: 为Dronpa -接头- Dronpa蛋白的光控模型。照明用400纳米的光将二聚化（和tetramerize）Dronpa并使其荧光。照明用500纳米的光将导致Dronpa蛋白，其中单体Dronpa是无荧光的解离。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54707/54707fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/54707/54707fig8.jpg" /> 图8: 使用LED热流量的Dronpa性能光控表征和f相结合<html>低流式细胞仪。 X轴上面所示和所述Dronpa平均荧光强度（MFI）描述了随着时间的推移细胞照亮。所述Dronpa - 接头Dronpa融合蛋白可使用设备（左）被photoswitched几次。在Dronpa荧光强度自然恢复只发生缓慢，效率低下（右）。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54707/54707fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图9" src="/files/ftp_upload/54707/54707fig9.jpg" /> 图9:光控Dronpa动力学分析。 Dronpa光控的动力学进行了计算的初始4分钟。照明和回收率的计算初始4分钟。由彩色区域指示在黑暗中孵育。每个计算的斜坡粗体数字描绘。ource.jove.com/files/ftp_upload/54707/54707fig9large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about LED Thermo Flow â€” Combining Optogenetics with Flow Cytometry at JoVE.com

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LED热流量 - 结合光遗传学与流式细胞仪

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

led-thermo-flow-combining-optogenetics-with-flow-cytometry

Universidad San Sebastian

Research

JoVE Journal

Biology

8.5K 次观看.  Max-Planck Institute for Immunobiology und Epigenetics. Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

视频: LED Thermo Flow — Combining Optogenetics with Flow Cytometry

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

科研

遗传学

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

免疫与感染

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

可靠地工程和控制哺乳动物细胞中稳定的光遗传学基因回路

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

生物工程

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

双分子荧光互补流式细胞仪分析：高通量的定量方法研究蛋白质 - 蛋白质相互作用

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

生物学

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

通过不同的均质化方法从小鼠大脑/脊髓中检索到的多种细胞类型的同步流细胞测量特征

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

医学

<meta charset="utf8"></head><body>与真菌伴侣共培养蔗糖分泌蓝细菌的方案

Assembly and Quantification of Co-Cultures Combining Heterotrophic Yeast with Phototrophic Sugar-Secreting Cyanobacteria

With the increasing demand for sustainable biotechnologies, mixed consortia containing a phototrophic microbe and heterotrophic partner species are being explored as a method for solar-driven bioproduction. One approach involves the use of CO2-fixing cyanobacteria that secrete organic carbon to support the metabolism of a co-cultivated heterotroph, which in turn transforms the carbon into higher-value goods or services. In this protocol, a technical description to assist the experimentalist in the establishment of a co-culture combining a sucrose-secreting cyanobacterial strain with a fungal partner(s), as represented by model yeast species, is provided. The protocol describes the key prerequisites for co-culture establishment: Defining the media composition, monitoring the growth characteristics of individual partners, and the analysis of mixed cultures with multiple species combined in the same growth vessel. Basic laboratory techniques for co-culture monitoring, including microscopy, cell counter, and single-cell flow cytometry, are summarized, and examples of nonproprietary software to use for data analysis of raw flow cytometry standard (FCS) files in line with FAIR (Findable, Accessible, Interoperable, Reusable) principles are provided. Finally, commentary on the bottlenecks and pitfalls frequently encountered when attempting to establish a co-culture with sugar-secreting cyanobacteria and a novel heterotrophic partner is included. This protocol provides a resource for researchers attempting to establish a new pair of co-cultured microbes that includes a cyanobacterium and a heterotrophic microbe.

<meta charset="utf8"></head><body>异养酵母与光养分泌糖的蓝细菌相结合的共培养物的组装和定量

This protocol provides a comprehensive guideline for the setup and quantitative monitoring of co-cultures, including photoautotrophic sugar-secreting cyanobacteria and heterotrophic yeasts.

异养酵母与光养分泌糖的蓝细菌相结合的共培养物的组装和定量

With the increasing demand for sustainable biotechnologies, mixed consortia containing a phototrophic microbe and heterotrophic partner species are being ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

遗传条形码与荧光蛋白的复用的应用程序

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

重组离子通道的相对细胞表面表达的总用流式细胞仪测定

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

LED热流量 - 结合光遗传学与流式细胞仪

摘要

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此视频中的章节

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

基于多色流式细胞仪的 t 细胞线粒体和溶酶体的定量

可靠地工程和控制哺乳动物细胞中稳定的光遗传学基因回路

双分子荧光互补流式细胞仪分析：高通量的定量方法研究蛋白质 - 蛋白质相互作用

通过不同的均质化方法从小鼠大脑/脊髓中检索到的多种细胞类型的同步流细胞测量特征

异养酵母与光养分泌糖的蓝细菌相结合的共培养物的组装和定量

异养酵母与光养分泌糖的蓝细菌相结合的共培养物的组装和定量

异养酵母与光养分泌糖的蓝细菌相结合的共培养物的组装和定量

遗传条形码与荧光蛋白的复用的应用程序

重组离子通道的相对细胞表面表达的总用流式细胞仪测定