这项研究是由一个自然科学和工程加拿大（NSERC）的研究资助局和阿尔伯塔省农业资金联盟授予DRB支持。 AMR是通过一个NSERC凡尼尔加拿大研究生奖学金，阿尔伯塔大学的助教和一个英国女王伊丽莎白二世研究生奖学金的支持。

执行此过程可在500μL的硅化管或6毫升聚苯乙烯圆底流式细胞仪管。后者通常是与其他程序一起进行可行性分析时使用。所有卷6毫升聚苯乙烯圆底流式细胞仪管。 500μL的硅化管，减少了1 / 5的所有卷。 流式细胞仪分析的最佳浓度为2-4 × 10 6细胞，每200μL体积。细胞的损失可能会导致此过程，因此，我们建议每个样本程序开始在4 × 10 6个细胞组成。 1。细胞制备<ol><li>收获细胞 - 指相应的细胞系或原电池隔离的具体程序。 </li><li>离心机在335 X克的样品为10分钟，倒出上清液。 </li><li>悬浮细胞在2毫升1 ×磷酸盐缓冲液（PBS） - / - （无钙，镁）。 </li><li>离心机在335 X克的样品为10分钟，倒出上清液。 </li><li>在1毫升1 × Annexin V的结合缓冲液重悬细胞。 </li><li>离心机在335 X克的样品为10分钟，倒出上清液。 </li><li> 100μL的1 × Annexin V的结合缓冲液中的悬浮细胞。 </li></ol> 2。应用膜联蛋白V / PI染色<ol><li>根据制造商的建议，加入Annexin V的[例如5μLAnnexin V的的Alexa Fluor 488（分子探针，A13201）]。 </li><li>在黑暗中为15分钟，在室温下孵育管。 </li><li>到每个反应管中加入100μL1 × Annexin V的结合缓冲液。应该有大约200μL每管。 </li><li>新增4μL的PI（Sigma公司，猫的P - 4864 - 10ML）已稀释在1 × Annexin V的结合缓冲液（即1μL9μL，1 × Annexin V的结合缓冲液PI）1:10。这将产生一个最终的PI浓度2微克/毫升每个样品。 </li><li>在黑暗中为15分钟，在室温下孵育管。 </li><li>加入500μL，1 × Annexin V的结合缓冲液洗细胞。 </li><li>离心机在335 X克的样品为10分钟，倒出上清液。 </li><li> 500μL的1 × Annexin V的结合缓冲液和500μL2％的甲醛，创建一个1％的甲醛（固定液）解决方案中的悬浮细胞。混合管轻轻弹。 </li><li>固定在10分钟的冰样本。此外，样品可以存储在一夜之间在4 ° C黑暗环境中。如果选择了后一种方法是，确保跨越用膜联蛋白V / PI（包括补偿控制）标记的所有管。 </li><li>加入1毫升1 × PBS -每个样品和混合轻轻弹- /。 </li><li> 425 X克离心管8分钟，倒出上清液。 </li><li>重复步骤2.10和2.11。 </li><li>悬浮沉淀弹管。 </li><li>加入16μL1:100稀释的RNA酶A（Sigma公司，R4642），给予终浓度为50微克/毫升。 15分钟在37 ° C。 </li><li>加入1毫升1 × PBS - / -混合，轻轻地弹。 </li><li> 425 X克八分钟离心管。 </li><li>要分析的样品现在已经准备好。此外，样品可以用于后续的染色步骤，如果膜联蛋白/ PI染色正在与其他程序的并行执行。 </li></ol> 3。代表性的成果: 如图1所示，有不同程度的假阳性PI染色的细胞类型和细胞系的例子。要考虑为假阳性事件，细胞固定，然后用RNase A的看待这个结果在假阳性事件的数量显着减少，细胞相比，没有经过核糖核酸治疗（图2B）。图2C显示行核糖核酸治疗的细胞，细胞没有核糖核酸治疗相比，代表图像。图3显示了如何与固定和RNase处理步骤修改后的膜联蛋白V / PI双协议，通过限制假阳性染色事件的数量，提高常规协议。 <img src="/files/ftp_upload/2597/2597fig1.jpg" alt="图1" /> 图1。传统的碘化丙啶染色协议导致大量的误报事件 。，我们曾评估跨越广泛的动物模型，以及在多种细胞系10假跨原代细胞的阳性染色程度。代表的图像显示在三个独特的细胞群（一个常用的细胞株 - RAW巨噬细胞和两个主要的细胞 - 小鼠骨髓巨噬细胞（BMM）的金鱼原发性肾脏巨噬细胞（PKM））的假阳性染色程度。真阳性事件显示核车厢内的预期之间的PI和DRAQ5的合作本地化（黄色区域描绘PI和DRAQ5之间的共定位）。 DRAQ5是高度membraNE渗透性染料，准确污渍核舱在活的和死的细胞 10,11,12 。为了方便视觉决心共定位DRAQ5染色绿色pseudocolour。 <img src="/files/ftp_upload/2597/2597fig2.jpg" alt="图2" /> 图2。提高PI核染色的准确性。 （一）我们评估的PI基于一个良好的特点核污渍的数量（BrdU，7 - AAD和DAPI）的相似性DRAQ5程度的核染色的准确性。 BrdU是一种人工合成的核苷纳入增殖细胞的DNA，因此只会弄脏核车厢内。 7 - AAD有一个高亲和力的DNA，类似为PI，不能跨越一个完整的质膜。的DAPI也有很强的亲和力的DNA，是最常用的核染色荧光显微镜。 BrdU，7 - AAD，DAPI染色和DRAQ5之间的相似程度&gt; 99％，突出自己的能力，准确地在RAW 264.7巨噬细胞的细胞核染色。相比之下，基于传统协议的细胞质PI染色％的染色相对DRAQ5相似的一个显着减少。 （二）观察到类似的结果在两个主要测试细胞:小鼠骨髓巨噬细胞（BMM）的金鱼原发性肾脏巨噬细胞（PKM）。 50微克/毫升核糖核酸酶在染色过程中特定阶段的一个团，消除非核PI染色，显着增加的相似程度相对DRAQ5染色。 （三）原发性肾脏巨噬细胞的具有代表性的图像显示为核糖核酸酶治疗和无细胞的相似程度。为了方便治疗之间的视觉差异的决心，DRAQ5绿色。黄色区域描绘BrdU和DRAQ5，有价证券和DRAQ5之间的共定位。 <img src="/files/ftp_upload/2597/2597fig3.jpg" alt="图3" /> 图3。改性膜联蛋白V / PI显着降低虚假凋亡/坏死事件。小金鱼肾巨噬细胞（PKM）染色与传统的和修改过的膜联蛋白V / PI协议。散点图描述膜联蛋白V / PI双染色金鱼的PKM。左边的散点图显示传统PKM的细胞的膜联蛋白V / PI染色（无核糖核酸）。在右边的散点图显示与修改后的膜联蛋白V / PI双协议（核糖核酸）染色PKM细胞。此外核糖核酸酶中的一个显着减少因假阳性污渍的去除PI染色的结果。 PKM的细胞，然后进行分析的基础上文化早期祖细胞内的不同人群（EP），单核细胞（单声道）和成熟的巨噬细胞（垫MAC）。积极PI事件的数量减少，由于去除误报事件是在大的成熟的巨噬细胞更明显比在较小的早期祖细胞。在传统的协议为基础的结论有误提出一个较为成熟的巨噬细胞亚群的细胞死亡中的积极的关系。

<ol>
	<li>Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+apoptosis.+Flow+cytometric+detection+of+phosphatidylserine+expression+on+early+apoptotic+cells+using+fluorescein+labeled+Annexin+V.">A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V.</a> J Immunol Methods. 184, 39-51 (1995).</li><li>Vermes, I., Haanen, C., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+of+apoptotic+cell+death.">Flow cytometry of apoptotic cell death.</a> J Immunol Methods. 243, 167-190 (2000).</li><li>Cornelissen, M., Philippe, J., De Sitter, S., De Ridder, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+V+expression+in+apoptotic+peripheral+blood+lymphocytes:+An+electron+microscopic+evaluation.">Annexin V expression in apoptotic peripheral blood lymphocytes: An electron microscopic evaluation.</a> Apoptosis. 7, 41-47 (2002).</li><li>Fried, J., Perez, A. G., Clarkson, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytofluorometric+analysis+of+cell+cycle+distributions+using+propidium+iodide.+Properties+of+the+method+and+mathematical+analysis+of+the+data.">Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data.</a> J Cell Biol. 71, 172-181 (1976).</li><li>Bacso, Z., Everson, R. B., Eliason, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+of+Annexin+V-binding+apoptotic+cells+is+highly+fragmented.">The DNA of Annexin V-binding apoptotic cells is highly fragmented.</a> Cancer Res. 60, 4623-4628 (2000).</li><li>Darzynkiewicz, Z., Bruno, S., Del Bino, G., Gorczyca, W., Hotz, M. A., Lassota, P., Traganos, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+of+apoptotic+cells+measured+by+flow+cytometry.">Features of apoptotic cells measured by flow cytometry.</a> Cytometry. 13, 795-808 (1992).</li><li>Kroemer, G., Dallaporta, B., Resche-Rigon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+death/life+regulator+in+apoptosis+and+necrosis.">The mitochondrial death/life regulator in apoptosis and necrosis.</a> Annu. Rev. Physiol. 60, 619-642 (1998).</li><li>Denecker, G., Vercammen, D., Declercq, W., Vandenabeele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+and+necrotic+cell+death+induced+by+death+domain+receptors.">Apoptotic and necrotic cell death induced by death domain receptors.</a> Cell Mol. Life Sci. 58, 356-370 (2001).</li><li>Faleiro, L., Lazebnik, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspases+disrupt+the+nuclear-cytoplasmic+barrier.">Caspases disrupt the nuclear-cytoplasmic barrier.</a> J Cell Biol. 151, 951-959 (2000).</li><li>Rieger, A. M., Hall, B. E., Luong, L. T., Schang, L. M., Barreda, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conventional+apoptosis+assays+using+propidium+iodide+generate+a+significant+number+of+false+positives+that+prevent+accurate+assessment+of+cell+death.">Conventional apoptosis assays using propidium iodide generate a significant number of false positives that prevent accurate assessment of cell death.</a> J Immunol Methods. 358, 81-92 (2010).</li><li>Edward, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+Use+of+DNA-specific+anthraquinone+dyes+to+directly+reveal+cytoplasmic+and+nuclear+boundaries+in+live+and+fixed+cells.">Minireview: Use of DNA-specific anthraquinone dyes to directly reveal cytoplasmic and nuclear boundaries in live and fixed cells.</a> Mol. Cells. 27, 391-396 (2009).</li><li>Njoh, K. L., Patterson, L. 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膜联蛋白V / PI双协议这里介绍的是常规协议的修改后的版本，并考虑到存在的RNA在细胞质中也有PI高亲和力。介绍RNA酶A（50微克/毫升）以下1％的甲醛固定在染色过程中的步骤，大大提高了核PI染色的准确性。核PI染色或细胞膜Annexin V的染色观察到任何负面影响。没有核糖核酸酶A治疗，高达40％的PI染色结果可能会导致误报事件，从而导致潜在的重大负面影响上下游结论10。 修改后的膜联蛋白V / PI染色协议很简单，在一个广泛的细胞类型（ 原代细胞已被用于有效的小鼠骨髓巨噬细胞和脾;猪肺居民细胞，肺泡巨噬细胞，肠系膜淋巴结分离，外周血白细胞脾;鸡胚细胞;金鱼原发性肾脏巨噬细胞;鲤鱼外周血白细胞;细胞株RAW 264.7巨噬，Jurkat T细胞，猪3D4/31巨噬，金鱼鱼翅纤维母细胞CCL - 71，3B11鲶鱼 B细胞10）。重要的是要注意假阳性PI染色，与原代细胞一般有虚假的10个阳性事件较多，影响细胞的差异。虚假的差异，这些细胞群之间的积极PI染色可部分归因于细胞大小的差异，但也可能会导致RNA含量的差异。因此，此过程纳入特别是有关的实验系统，利用，等等，细胞遗传毒性应激，细胞与细胞周期逮捕胸苷或羟基脲等药物，病毒感染的细胞，对胚胎干细胞里发育进程的研究治疗特点是RNA的合成离散变化。

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<td>1 x PBS-/-</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>1 x Annexin V Binding Buffer</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>556454</td>
<td>&nbsp;</td>
<tr>
<td>Annexin V-Alexa Fluor 488</td>
<td>&nbsp;</td>
<td>Molecular Probes (Invitrogen)</td>
<td>A13201</td>
<td>&nbsp;</td>
<tr>
<td>Propidium iodide (PI)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P4864</td>
<td>1 mg/mL in H2O</td>
</tr>
<tr>
<td>2% Formaldehyde</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F1268</td>
<td>&nbsp;</td>
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<td>RNase A from bovine pancreas</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>R4642</td>
<td>&nbsp;</td>
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<td>DRAQ5</td>
<td>&nbsp;</td>
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<td>DR50050</td>
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modified annexin v/propidium iodide apoptosis assay for accurate assessment of cell death

细胞凋亡的研究有了显着的影响以来，流式细胞仪为基础的方法的引进。碘化丙啶（PI）是广泛用于与Annexin V的结合来确定，如果细胞通过质膜的完整性和通透性1,2差异可行的，凋亡，或坏死。膜联蛋白V / PI协议是一个常用的方法，为研究细胞凋亡3。 PI是，往往比其他核污渍，因为它是经济，稳定和细胞活力的一个良好指标，根据其能力，以排除在活细胞4,5染料。 PI的进入细胞的能力是依赖于细胞膜的通透性; PI不染色居住或早期由于存在一个完整的质膜 1,2,6的细胞凋亡。晚期凋亡和坏死细胞，血浆和核膜的完整性下降 7,8，使PI传递透过膜，插成核酸，并显示红色荧光 1,2,9 。不幸的是，我们发现，传统的膜联蛋白V / PI双协议导致大量的误报事件（高达40％），这是与PI染色的RNA 在细胞质车厢10。原代细胞和细胞株在动物模型的广泛影响，大细胞:显示最高的发生10（核细胞质比率&lt;0.5 ）。在这里，我们展示了修改后的膜联蛋白V / PI方法，提供了一个评估与常规方法相比，细胞死亡的显着改善。该协议需要在修复细胞，促进一成以下的染色细胞的RNase进入细胞通透性的变化。的时间和浓度的RNase一个已经优化去除细胞质RNA。结果是比传统的膜联蛋白V / PI协议（&lt;5％，与细胞质PI染色的事件）的显着改善。

Watch this Scientific Journal Video about Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death at JoVE.com

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

为评估细胞死亡的一种准确的方法是描述。该协议改进传统的膜联蛋白V /碘化丙碘化物（PI）的协议，这显示多达40％的假阳性的细胞株和原代细胞的动物模型的广泛事件。

改性膜联蛋白V /碘化丙啶检测细胞凋亡细胞死亡的准确评估

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

modified-annexin-vpropidium-iodide-apoptosis-assay-for-accurate

Nanyang Technological University

Research

JoVE Journal

Biology

102.9K 次观看.  University of Alberta. 为评估细胞死亡的一种准确的方法是描述。该协议改进传统的膜联蛋白V /碘化丙碘化物（PI）的协议，这显示多达40％的假阳性的细胞株和原代细胞的动物模型的广泛事件。

视频: Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

焦磷酸测序：一个准确的基因分型的简单方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

科研

生物学

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

专访：干细胞研究的生物反应器和三维支架材料表面改性

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

基因小鼠胚胎干细胞的基础含量为心源性诱导效率量化

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

A &#946;-glucuronidase (GUS) Based Cell Death Assay

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes.

A &#946;-glucuronidase GUS Based Cell Death Assay

Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.

一个β-葡萄糖醛酸酶（GUS）的基细胞死亡含量

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

使用LysoTracker检测及鉴别胚胎干细胞在胚胎细胞程序性死亡

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

E4orf4诱导的细胞死亡的击倒效果的基因表达和测量条件击倒细胞系的制备

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像<em&gt;果蝇</em&gt;感光细胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

策略跟踪Anastasis，A细胞存活的现象，细胞凋亡反转

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

改性膜联蛋白V /碘化丙啶检测细胞凋亡细胞死亡的准确评估

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