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。

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	<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. 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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。

<table><tbody><tr><td>1 x PBS-/-</td><td></td><td></td><td></td></tr><tr><td>1 x Annexin V Binding Buffer</td><td>BD Biosciences</td><td>556454</td><td></td></tr><tr><td>Annexin V-Alexa Fluor 488</td><td>Molecular Probes, Life Technologies</td><td>A13201</td><td></td></tr><tr><td>Propidium iodide (PI)</td><td>Sigma-Aldrich</td><td>P4864</td><td>1 mg/mL in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>2% Formaldehyde</td><td>Sigma-Aldrich</td><td>F1268</td><td></td></tr><tr><td>RNase A from bovine pancreas</td><td>Sigma-Aldrich</td><td>R4642</td><td></td></tr><tr><td>DRAQ5</td><td>Biostatus</td><td>DR50050</td><td>Dilute 1:60 in 1 x PBS-/-</td></tr></tbody></table>

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

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

Detection and Isolation of Apoptotic Bodies to High Purity

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. It has been proposed that ApoBDs can aid cell clearance as well as intercellular communication through trafficking biomolecules. Conventional approaches used for the identification and isolation of ApoBDs are often limited by the lack of accurate quantification and low sample purity. Here, we describe a workflow to confirm the induction of apoptosis, validate ApoBD formation, and isolate ApoBDs to high purity. We will also outline and compare fluorescence-activated cell sorting (FACS) and differential centrifugation based approaches to isolate ApoBDs. Furthermore, the purity of isolated ApoBDs will be confirmed using a previously establish flow cytometry-based staining and analytical method. Taken together, using the described approach, THP-1 monocyte apoptosis and apoptotic cell disassembly was induced and validated, and ApoBD generated from THP-1 monocytes were isolated to a purity of 97-99%.

A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity.

高纯度凋亡体的检测与分离

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

科研

生物化学

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

监测劈开caspase - 3活性和钾诱导膜去极化使用活细胞成像和Cleaveable荧光染料基板的永生的少突胶质细胞凋亡

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

神经科学

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.

The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells&#39; mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes &#8211; Hoechst 33342 and propidium iodide.

自动差分核染色测定，准确测定三元细胞毒性

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes ...

癌症研究

Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V and mitochondrial membrane potential) and late biomarkers (caspases 3 and 7, and DNA damage) of apoptosis were measured in experimental and control cultures. Cultures were incubated for 24 hours in a humidified incubator at 37 &#176;C with 5% CO2. The cells were then detached using trypsin and enumerated using a flow cytometric cell count assay. Cells were further analyzed for apoptosis using an Annexin V assay, a mitochondrial electrochemical transmembrane potential assay, a caspase 3/7 assay, and a DNA damage assay. This article provides an overview of apoptosis and traditional flow cytometry, and elaborates flow cytometric protocols for processing and analyzing SiHa cells. The results describe positive, negative, and sub-optimal experimental data. Also discussed are interpretation and caveats in performing flow cytometric analysis of apoptosis using this analytical platform. Flow cytometric analysis provides an accurate measurement of early and late biomarkers for apoptosis.

Apoptosis can be characterized by flow cytometric analysis of early and late apoptotic biomarkers. The cervical cancer cell line, SiHa, was analyzed for apoptosis biomarkers after treatment with Actinomycin D using a benchtop flow cytometer.

放线菌素D处理SiHa宫颈癌细胞中凋亡生物标志物的流式细胞术分析

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V ...

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

使用胱天蛋白酶复分析，以确定细胞凋亡的下丘脑细胞模型

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

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 ...

生物学

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

活性检测在培养细胞

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

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 ...

An Introduction to Cell Death

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as cell injury, low nutrient levels, or signaling proteins. Whereas necrosis is considered to be an &#8220;accidental&#8221; or unexpected form of cell death, evidence exists that apoptosis and autophagy are both programmed and &#8220;planned&#8221; by cells.In this introductory video, JoVE highlights key discoveries pertaining to cell death, including recent work done in worms that helped identify genes involved in apoptosis. We then explore questions asked by scientists studying cell death, some of which look at different death pathways and their interactions. Finally, several methods to assess cell death are discussed, and we note how researchers are applying these techniques in their experiments today.

Cell Death: Apoptosis, Autophagy and Necrosis

导论细胞死亡

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as ...

教育

JoVE Science Education

高级生物学

细胞生物学

Annexin V and Propidium Iodide Labeling

Staining with annexin V and propidium iodide (PI) provides researchers with a way to identify different types of cell death&#8212;either necrosis or apoptosis. This technique relies on two components. The first, annexin V, is a protein that binds certain phospholipids called phosphatidylserines, which normally occur only in the inner, cytoplasm-facing leaflet of a cell&#8217;s membrane, but become &#8220;flipped&#8221; to the outer leaflet during the early stages of apoptosis. The second component is the DNA-binding dye molecule PI, which can only enter cells when their membranes are ruptured&#8212;a characteristic of both necrosis and late apoptosis.This video article begins with a review of the concepts behind annexin V and PI staining, and emphasizes how differential patterns of staining can be used to distinguish between cells progressing down different death pathways. We then review a generalized protocol for this technique, followed by a description of how researchers are currently using annexin V and PI staining to better understand cell death.

Cell Death Pathways and Annexin V &amp; PI Labeling studies

膜联蛋白 V 和碘化丙啶标签

Staining with annexin V and propidium iodide (PI) provides researchers with a way to identify different types of cell death&#8212;either necrosis or ...

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

摘要

探索更多视频

此视频中的章节

高纯度凋亡体的检测与分离

监测劈开caspase - 3活性和钾诱导膜去极化使用活细胞成像和Cleaveable荧光染料基板的永生的少突胶质细胞凋亡

自动差分核染色测定，准确测定三元细胞毒性

放线菌素D处理SiHa宫颈癌细胞中凋亡生物标志物的流式细胞术分析

使用胱天蛋白酶复分析，以确定细胞凋亡的下丘脑细胞模型

评估肾细胞蛋白激酶C同工酶，线粒体功能及细胞活力的

活性检测在培养细胞

策略跟踪Anastasis，A细胞存活的现象，细胞凋亡反转

导论细胞死亡

膜联蛋白 V 和碘化丙啶标签

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

摘要

探索更多视频

此视频中的章节

高纯度凋亡体的检测与分离

监测劈开caspase - 3活性和钾诱导膜去极化使用活细胞成像和Cleaveable荧光染料基板的永生的少突胶质细胞凋亡

自动差分核染色测定，准确测定三元细胞毒性

放线菌素D处理SiHa宫颈癌细胞中凋亡生物标志物的流式细胞术分析

使用胱天蛋白酶复分析，以确定细胞凋亡的下丘脑细胞模型

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

活性检测在培养细胞

策略跟踪Anastasis，A细胞存活的现象，细胞凋亡反转

导论细胞死亡

膜联蛋白 V 和碘化丙啶标签

评估肾细胞蛋白激酶C同工酶，线粒体功能及细胞活力的