1。大里可行性套件 - 死细胞红试剂染色细胞<ol><li>此过程开始与U2OS细胞（美国典型培养物保藏）已转用CellLight核- GFP * BacMam 2.0 *. （注:细胞也可以降速，并在培养基或PBS重悬）。 </li><li>与PI染色死细胞，100μL的细胞转移到一个新的离心管。需要注意的是1 × 10 5 1 × 10 7细胞/ ml的浓度是这个实验的建议，虽然浓度不准确。 </li><li>下一步，染色的死细胞，加入1μLPI为基础的大里死细胞红试剂。涡简单地混合。孵育室温在黑暗时间为1-5分钟的大里死细胞红试剂和细胞混合物。 </li><li>孵化后，立即进行的大里基于图像流式细胞仪分析样品。 </li></ol> 2。使用执行细胞活性测定大里图像基于流式细胞仪<ol><li>大里流式细胞仪使用的一次性塑料大里细胞分析幻灯片，特别适合到流式细胞仪，可容纳在两个单独的封闭商会（A和B）25μL样品。处理幻灯片时，一定要始终坚持由双方。 </li><li>慢慢进入半月亮形的样品装卸区要加载的幻灯片，移液器25μL的样品，照顾，以避免形成气泡或回飞溅。不要超过或低于填写。 </li><li>在这里，控制细胞（未染色，非转）装在室A和染绿色荧光蛋白表达细胞室B.加载控制样本将有助于分析数据时确定的自体荧光的水平。 </li><li>要执行一个可行性测定的流式细胞仪的主屏幕上触摸GFP / RFP。检测选项就会出现在右侧。选择绿色荧光蛋白"+"生存能力"。 </li><li>下一步，名称的样本序列， 立即按名称 </strong&gt;。然后，使用触摸屏，键入样品系列的名称，然后按保存。 （注: 选择名称 ，自动分配每个样品系列的日期和时间的名称。 ） </li><li>命名后的样品，大里流式细胞仪将自动进入测量屏幕。按测量屏幕的右下角一半" 背景 "选项卡。 </li><li>接下来，与对照样本（一）面临从流式细胞仪，插入幻灯片在装货港，直到它停止。不要使用武力。 </li><li>在背景屏幕，触摸按插入新的未染色细胞控制。幻灯片将自动被拉进流式细胞仪和一个细胞的实时图像将显示在屏幕上。 </li><li>使用对焦旋钮，把适当的平面细胞。细胞应均匀黑暗与每一个细胞周围的耀眼光环（图1左）。 </li><li>细胞s可能会undercounted时的背景和细胞边缘之间的过渡是模糊的，使细胞界限没有明确界定（图1，中心）。细胞可能overcounted当他们有明亮的中心和黑暗的周长（图1右）。 </li><li>为了更好地观察细胞的人口，同时重点，幻灯片的红色变焦指示灯按钮为4X或16X 。 </li><li>一旦细胞已成为关注的焦点带来的， 触摸按运行未染色细胞控制措施的背景荧光。 </li><li>流式细胞仪将自动采集和分析样品的图像。这大约需要1分钟默认（9图像）模式图像捕获和分析。 </li><li>每个视野的缩略图显示，因为它们是捕获和进度条提供了一个持续的更新。图像采集完成后，流式细胞仪自动弹出的幻灯片，拍摄的图像进行分析，并提供数据。 </li><li>存储的数据将显示在下拉菜单的" 背景 "选项卡上流式细胞仪下降。背景对照样品将被分配名称相同的实验，将在下拉菜单中进口。注:如果是无法运行的背景下控制，流式细胞仪会自动分配一个荧光阈值。这可以手动更改后执行检测。 </li><li>运行PI染色转样本，从流式细胞仪中删除幻灯片，翻转它重新插入转样品面临从仪器。 </li><li>按样本选项卡，然后触摸按插入新样品 。当图像出现在屏幕上，使用图像调整旋钮，使细胞像以前那样成为关注的焦点。然后触摸按运行示例 。 </li><li>图像采集完成后，出现在样品标签和流式细胞仪自动分析数据盟友退出幻灯片。 </li><li>妥善处理大里细胞作为生物危险废物分析幻灯片。大里细胞分析幻灯片不能重复使用。 </li></ol> 3。设置阈值和盖茨<ol><li>一旦样品运行，阈值现在可以适用于家样本在一个特定的细胞群。在这里，我们将调整阈值来确定的活细胞和死细胞百分比GFP的细胞。 </li><li>根据样品标签，表达GFP的细胞，活的和死的细胞表达绿色荧光蛋白，总的生存能力，平均细胞大小，计算细胞数量，细胞浓度的数量和百 ​​分比显示。 </li><li>此外，直方图显示细胞的大小，绿色荧光和红色荧光（PI）的图显示在屏幕的底部。 </li><li>若要设置单元格的大小门，触摸细胞大小的缩略图，打开细胞大小的直方图。虽然培养的细胞中很少有Debris，其他样品可能含有大于或小于细胞的碎片。 </li><li>为了排除这种碎片，滑动的滑块上的蓝色按钮，排除大型和小型的事件。然后，触摸应用的数据重新分析和更新的结果。 </li><li>接下来，添加GFP荧光分析，触摸GFP直方图缩略图来打开它。滑动蓝色栏设置的阈值，只是权利的最暗的高峰，为指导的对照样品。 </li><li>这使得分析，包括GFP阳性细胞，但不包括autofluorescent细胞。自体荧光经常观察到细胞内生物分子很高兴。 </li><li>触摸应用 ，以确认并返回到前一个画面。 样品标签下所显示的数据，现在应该反映两个荧光通道的大门，设置阈值。 </li><li>下一步，要分开PI染色细胞的自体荧光，按T他PI直方图缩略图打开PI的直方图。 </li><li>同样，控制样品峰将显示为一个直方图的灰色高峰。红峰是样品的荧光。背景荧光显示流式细胞仪0 RFU值最接近的一个高峰。 </li><li>为了消除在P​​I通道的背景荧光，移动滑块上的蓝色按钮来调整阈值排除的高峰，代表背景荧光，从控制样本作为参考使用灰色的高峰。触摸应用 ，以确认并返回到前一个画面。 </li><li>接下来，直观地确认每一个细胞都正确分配，按缩放 "选项卡中的图像的缩略图中选择捕获领域的审查，然后按"图层"选项卡。 </li><li>接下来，按GFP或PI按钮，以显示拍摄的图像通过特定渠道。再次按下相同的按钮，删除显示层;或叠加层，触摸多层按钮。 </li><li>要确定如何通过计算一个特定的通道，触摸圈的细胞。通过只有亮场的通道，不表达荧光将盘旋在蓝色计数细胞。这表明，特定的细胞是活的（即有价证券负）。 </li><li>将出现在细胞表达绿色荧光蛋白GFP的通道，将在绿色盘旋。死细胞与死细胞红染，将在PI通道，将在红色圆圈。 </li><li> ，在红色和绿色通道的计数细胞将黄色盘旋，这表明细胞表达GFP，但也与PI染色，因此是死的。 </li><li>偶尔，黑色圆圈会出现在屏幕上，这些都是已经确定的对象，但已经从基于细胞大小的分析排除。 </li><li>继续到FINE调整使用刚才所描述的，用适当的颜色盘旋，直到每个细胞表达荧光的步骤上的荧光直方图的阈值。 </li><li>所有分析参数自动保存数据"选项卡下。大里流式细胞仪可以存储大约500个样品运行的数据文件。 </li><li>在数据选项卡中存储的每个文件再分析。通过选择" 数据 "选项卡文件将被打开，所有直方图和层变得活跃。 </li><li>存档数据，并生成报告，在流式细胞仪中存储的数据可能被转移到一台电脑使用USB驱动器。 </li></ol> 4。代表性的成果: 大里图片基于流式细胞仪是用来测量荧光蛋白在细胞中的表达水平与核针对性GFP BacMam 2.0表达式构造转。四个有代表性的细胞类型，转（U2OS，293MSR，HEKn和CHO - S）。对于这些细胞株，表达GFP的细胞数据报道大里流式细胞仪和流式细胞仪分析同一样品的数据相比，。此外，细胞被染成红色的死细胞试剂的使用大里可行性套件上大里流式细胞仪和流式细胞仪，以确定死者的细胞群。 图2中的图形表示显示的不同的细胞类型，表达GFP的总人口的百分之。这些数据表明，大里流式细胞仪产生的数据具有可比性，以流式细胞仪产生的数据。 <img alt="图1" src="/files/ftp_upload/3659/3659fig1.jpg" /> 图1。细胞的重点应妥善分析前 。适当的分析（建议1 × 10 5到1 × 10 7细胞）与细胞浓度大里细胞分析幻灯片（左）在重点细胞均匀ð方舟整个细胞与周围每个细胞（中心）细胞的耀眼光环，可能undercounted时之间的背景和细胞边缘的过渡是模糊和细胞有不确定的边界（右）细胞也许overcounted当他们有明亮中心和黑暗的周长。 <img alt="图2" src="/files/ftp_upload/3659/3659fig2.jpg" /> 图2。从大里基于图像流式细胞仪荧光蛋白表达数据与使用流式细胞仪获得一个4在活细胞不同的细胞系的结果计算GFP表达侧方比较。

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Krissy Remple和Laurel石材生产在这篇文章中使用的仪器生命科技的员工。生命科技公司赞助制作这个视频文章。

刚才我们详细介绍了程序执行的可行性计数和评估GFP的表达，使用大里图像基于流式细胞仪。流式细胞仪是使用相同的过程，能够执行其他一些分析，包括:使用非转染细胞的凋亡，RFP和细胞活力分析。 荧光检测细胞活力，基因表达和细胞凋亡已成为在生物医学研究7-12中的关键方法。在过去，独立的仪器已用于获取有关细胞的数量和视觉信息。虽然已通过流式细胞仪获取有关细胞内异构人口的定量信息，视觉或定性的信息已经收集通过使用荧光显微镜 1-4 。在这里，我们提出了一种技术，桥梁的人口和单个细胞研究之间的差距。使用的大里基于图像流式细胞仪，定量DATA和视觉细胞的数据，对单个细胞或细胞群，可同时收集。大里仪器可用于一系列的应用，包括评估的基因表达，细胞凋亡检测，药物疗效的研究，并评估疾病进展。 大里图片基于流式细胞仪捕获和分析明场，红色荧光和绿色荧光图像，允许获得有关细胞亚群的信息。例如，在这个实验中，我们能够区分活细胞内表达GFP的细胞死细胞红色染料人口的死细胞。这使得在可行的细胞表达GFP的测量精度，并确定了人口下游加工中使用的细胞。重要的是要注意，在人口中的死细胞可以来自各种来源，包括在文化的正常细胞死亡或细胞死亡造成的环境条件（例如，胰蛋白酶或ATH选择的方法转染/转导致）。 在这里，我们展示了与U2OS细胞大里流式细胞仪的使用。类似的方法可以使用​​大多数哺乳动物和昆虫细胞中。对于每一个细胞系，大里流式细胞仪定量GFP表达的数据，这是没有可能与使用标准的荧光显微镜下细胞的视觉检查。虽然这些结果与流式细胞仪，他们是在短的时间内收集。流式细胞仪是能够准确计数细胞，直径5微米至60微米之间，最好在1 × 10 5到1 × 10 7细胞/毫升的浓度。 重要的是要指出，最标准的染色协议，包括死细胞红染色这里描述的协议，使用碘化丙啶区分活细胞和死细胞。这是一个挑战，在评估已为ST透细胞的可行性癌宁细胞内的标记，由于PI染色细胞与任何一个妥协的膜。由于任何与绿色或红色荧光通道，可检测的荧光染料或蛋白质可以的大里基于图像流式细胞仪分析，今后的研究可以探索使用替代染料，如胺反应的可行性染料 4 。一个Perfetto 等 2006年的一项研究12发现这些染料在歧视活的和死的细胞群易于使用和可重复性。 请注意，大里尊重它可以执行的分析类型的限制。例如，由于仪器使用的目标是4X，是有限的计数操作较大的细胞类型:如线粒体或囊泡的亚细胞结构，细菌细胞不能量化。还要注意，在大里的检测渠道是有限的，将激发和发射的渠道内的荧光团。虽然YFP和明亮mCherry信号C一个被检测，调光器mCherry信号不能。最后，大里只分析悬浮细胞。它将无法准确计数细胞坚持一个幻灯片。此外，虽然目前仅用于计数细胞形状不规则，包括一些酵母和原代细胞的能力有限。 此外，未来的研究将有可能解决这个系统的使用在评估细胞内标记的表达。个人染料，可以检查与相邻通道的光谱重叠使用Invitrogen公司的在线光谱查看<a href="http://www.invitrogen.com/spectraviewer">工具（www.invitrogen.com /</a>光谱查看）。由于这项技术的新颖性，全方位的应用还没有被发现。 总之，这里展示的大里基于图像流式细胞仪提出了一种新的技术，允许细胞群的同步可视化和分析，使评估单个细胞，以及为CEL升的人群在一个紧凑的格式用一个简单的工作流程。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>大里图像基于流式细胞仪</td><td> Invitrogen公司</td><td> T10796 </td></tr><tr><td>大里细胞分析幻灯片，50张幻灯片。 </td><td> Invitrogen公司</td><td> T10794 </td></tr><tr><td>大里细胞分析幻灯片，500幻灯片</td><td> Invitrogen公司</td><td> T10795 </td></tr><tr><td>大里可行性套件 - 死细胞红</td><td> Invitrogen公司</td><td> A10786 </td></tr><tr><td>大里可行性套件 - 死细胞绿</td><td> Invitrogen公司</td><td> A10787 </td></tr><tr><td>大里凋亡试剂盒</td><td> Invitrogen公司</td><td> A10788 </td></tr><tr><td> U2OS细胞</td><td></td><td></td></tr></tbody></table>

assessment of gfp expression and viability using the tali image-based cytometer

单细胞和人口信息通常获得通过流式细胞仪或荧光显微镜。然而，这两种方法提供不同的信息。流式细胞仪提供了定量的物理特性和染色或表达的多参数的信息，但不允许进行可视化。单机荧光显微镜提供可视化的数据，但不允许为简单的定量测量。 基于图像流式细胞仪桥梁这两种方法之间的差距， 使 2异质种群的快速可视化和成千上万的细胞同时定量分析。在这里，我们目前使用基于图像的大里3流式细胞仪进行细胞活力和绿色荧光蛋白（GFP）的表达分析的方法。大里仪器是一个3通道（红色荧光的绿色荧光，明场，）台式检测平台，offeRS通过流式细胞仪和荧光显微镜的几个优点。大里流式细胞仪是成本较低，占用的工作台空间少，需要较少的维护和工作流程已简化，使操作和分析变得更简单快捷。大里流式细胞仪是能够执行悬浮细胞检测，包括GFP和红色荧光蛋白（RFP）的表达，细胞凋亡 4-6和细胞与可行性分析碘化丙啶（PI） 的 7-11的范围。 在这里，我们展示了大里仪器的使用，在执行中表达GFP的细胞的细胞活力检测。使用大里可行性套件 - 死细胞红染GFP的细胞。细胞，然后吸管成大里细胞分析幻灯片和装入的流式细胞仪。明场，红色荧光和绿色荧光图像被捕获并使用特定的算法检测分析。然后生成直方图显示细胞大小，有价证券荧光荧光强度，和GFP荧光强度。然后将这些参数阈值到家庭，在一个特定的细胞群。 由一个侧面表明，这两种方法提供方的大里基于图像流式细胞仪和传统的流式细胞仪比较，细胞活力和蛋白表达的可比数据。但是，大里仪器提供额外的视觉信息不能使用流式细胞仪获得的细胞群。

Watch this Scientific Journal Video about Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer at JoVE.com

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

本协议描述了如何使用的大里基于图像流式细胞仪进行细胞活力和荧光表达分析。

GFP的表达和使用的大里基于图像流式细胞仪的可行性评估

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide ...

assessment-gfp-expression-viability-using-tali-image-based

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

20.9K 次观看.  Life Technologies. 本协议描述了如何使用的大里基于图像流式细胞仪进行细胞活力和荧光表达分析。

视频: Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.

The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is, discussed. Hair follicle delivery systems are described, such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.

有针对性的表达绿色荧光蛋白在头发上使用体外病毒转导的卵泡

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft ...

科研

生物学

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

计数和确定培养细胞的可行性

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Flow cytometry has been widely used to obtain information about DNA content in a population of cells, to infer relative percentages in different cell cycle phases. This technique has been successfully extended to the mitotic tissues of the model organism Drosophila melanogaster for genetic studies of cell cycle regulation in vivo. When coupled with cell-type specific fluorescent protein expression and genetic manipulations, one can obtain detailed information about effects on cell number, cell size and cell cycle phasing in vivo. However this live-cell method has relied on the use of the cell permeable Hoechst 33342 DNA-intercalating dye, limiting users to flow cytometers equipped with a UV laser. We have modified this protocol to use a newer live-cell DNA dye, Vybrant DyeCycle Violet, compatible with the more common violet 405nm laser. The protocol presented here allows for efficient cell cycle analysis coupled with cell type, relative cell size and cell number information, in a variety of Drosophila tissues. This protocol extends the useful cell cycle analysis technique for live Drosophila tissues to a small benchtop analyzer, the Attune Acoustic Focusing Cytometer, which can be run and maintained on a single-lab scale.

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo.

活细胞周期分析<em&gt;果蝇</em&gt;组织使用同调声聚焦流式细胞仪Vybrant的DyeCycle紫DNA染色

Flow cytometry has  been widely used to obtain information about DNA content in a population of  cells, to infer relative percentages in different cell ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

代谢率的测量<em&gt;果蝇</em&gt;使用呼吸运动

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the sequential cleavage of Amyloid Precursor Protein (APP) by &#946; and &#947;-secretases. Despite the importance of A&#946; to AD pathology, the subcellular localization of these cleavages is not well established. Work in our laboratory and others implicate the endosomal/lysosomal system in APP processing after internalization from the cell surface. However, the intracellular trafficking of APP is relatively understudied.
While cell-surface proteins are amendable to many labeling techniques, there are no simple methods for following the trafficking of membrane proteins from the Golgi. To this end, we created APP constructs that were tagged with photo-activatable GFP (paGFP) at the C-terminus. After synthesis, paGFP has low basal fluorescence, but it can be stimulated with 413 nm light to produce a strong, stable green fluorescence. By using the Golgi marker Galactosyl transferase coupled to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to accurately photoactivate APP in the trans-Golgi network. Photo-activated APP-paGFP can then be followed as it traffics to downstream compartments identified with fluorescently tagged compartment marker proteins for the early endosome (Rab5), the late endosome (Rab9) and the lysosome (LAMP1). Furthermore, using inhibitors to APP processing including chloroquine or the &#947;-secretase inhibitor L685, 458, we are able to perform pulse-chase experiments to examine the processing of APP in single cells.
We find that a large fraction of APP moves rapidly to the lysosome without appearing at the cell surface, and is then cleared from the lysosome by secretase-like cleavages. This technique demonstrates the utility of paGFP for following the trafficking and processing of intracellular proteins from the Golgi to downstream compartments.

While the transport of cell surface proteins is relatively easily studied, visualizing the trafficking of intracellular proteins is much more difficult. Here, we use constructs incorporating photoactivatable GFP and demonstrate a method to accurately follow the amyloid precursor protein from the Golgi apparatus to down-stream compartments and follow its clearance.

成像APP的细胞内运输与光活化GFP

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the ...

GFP的表达和使用的大里基于图像流式细胞仪的可行性评估

摘要

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有针对性的表达绿色荧光蛋白在头发上使用体外病毒转导的卵泡

计数和确定培养细胞的可行性

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

在体外重组活跃T。谷盗端粒酶

评估肾细胞蛋白激酶C同工酶，线粒体功能及细胞活力的

活细胞周期分析

代谢率的测量

成像APP的细胞内运输与光活化GFP

GFP的表达和使用的大里基于图像流式细胞仪的可行性评估

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