这项工作是支持的补助金从国家科学与工程研究理事会，加拿大，西飞和AFP

1。萃取材料的制备<ol><li>制备细胞<ol><li>种子组织培养细胞对实验前至少一天的酸处理过的盖玻片。我们用COS-7或U2OS​​，这两个细胞系分泌的蛋白质，具有强大的生产，有一个良好定义的ER。请注意，以实现高的提取效率，细胞应不超过80％汇合的盖玻片上的实验当天。 </li><li>如果一个特定的外源基因是被调查，细胞播种与使用标准转染协议感兴趣的含有该基因的质粒转染后一天。的细胞，然后使萃取前至少18小时的mRNA表达。 </li></ol></li><li>转换酶抑制剂治疗的细胞<ol><li>为了测试效果的ER基因与维护完整的核糖体，细胞是可以治疗的翻译抑制剂，破坏协会的核糖体与mRNA的11。 </li><li>抑制剂的翻译起始，如高三尖杉酯碱（HHT），pactamycin，或2 - （4 -甲基-2 ,6-dinitroanilino） - N-甲基丙酰胺（MDMP），防止与转录相关联的新的核糖体，同时允许从事核糖体完成翻译12-14。由于大多数蛋白质在哺乳动物细胞中的翻译率是每秒5个氨基酸，不管密码子使用或mRNA长度为15，在30分钟的处理应该清除核糖体关闭的消息的开放阅读框架，只要27000个核苷酸（编码的蛋白质只要为9000个氨基酸）。 </li><li>嘌呤酶抑制剂，如促进过早排出的新生肽链，从而破坏核糖体12。然而，为了完全破坏嘌呤霉素处理的核糖体与mRNA的关联，必须被添加到镁的螯合剂如EDTA，毛地黄皂苷提取缓冲液11。 </li> &lt;li&gt;在本实验中，将细胞与200μM，嘌呤霉素，5μMHHT，或控制介质的前30分钟提取处理。 </li></ol></li><li>毛地黄皂苷提取缓冲液的制备。可视化ER-本地化的mRNA无阻碍的自由（ 即细胞质，非ER相关的）成绩单，我们选择性地通透的细胞与毛地黄皂苷，甾体苷优先与3β-hydroxysterols的交互。在低浓度时，毛地黄皂苷选择性permeabilizes质膜的部分，导致&#39;泄漏&#39;16。与此相反，在ER膜和核包络，这是差的胆固醇，被原封不动16,17。 <ol><li>毛地黄皂苷（Sigma Aldrich公司）的粉末溶解于蒸馏水中，在5％重量/体积，此原液成小卷等分并存储在-20℃下根据我们的经验，多个冻结 - 解冻周期，降低溶解的毛地黄皂苷的效率。 </li><li> A股解决方案10倍的CHO缓冲液（1×工作溶液的浓度:115毫摩尔醋酸钾，pH为7.4的25mM HEPES，2.5毫摩尔MgCl 2，2mM的EGTA和150mM蔗糖）制备用无RNA酶的试剂，并储存于-20℃下的A工作液（1X CHO缓冲）是由无RNase水稀释的原液，可以存储在4°C </li></ol></li></ol> 2。毛地黄皂苷的提取<ol><li>准备提取解决方案<ol><li>平坦的表面上的加热块与被预加热至40℃并保持在此温度下提取期间。 </li><li>以形成平坦的工作表面是无RNase，加热块蘸有一些水和重叠有一块新鲜的:石蜡，M（比米斯公司，Inc制造）。石蜡片和加热块之间的空气泡被平滑使用无RNase的手套和kimwipes（VWR）。 </li><li> 1倍的CHO缓冲液温热至37℃，通过在水浴中孵育。 </li><li> 6孔培养板用于洗修复的细胞。 3孔的每行被用于一个盖滑。到第一行中的两口井，2毫升温热的CHO缓冲液中的溶液。到最后一个孔中，添加2毫升的固定溶液（PBS + 4％多聚甲醛（电子显微镜版））。此托盘可以被存储在37℃培养箱中培养，直到细胞提取准备。 </li><li>毛地黄皂苷的股票溶液稀释至0.025％，与温暖的CHO缓冲之前使用毛地黄皂苷提取溶液的制备方法。再次注意，嘌呤霉素处理的细胞中，在提取缓冲液必须另外含有的20mM EDTA，有效地破坏核糖体11。紧接之前提取的提取溶液滴加入100μl置于加热块上的封口膜覆盖。 </li></ol></li><li>毛地黄皂苷的提取和细胞固定<ol><li>从37℃的培养箱中取出的细胞。 </li><li>在提取过程中，盖玻片jewler的钳子的帮助操纵。同的钳子拿起盖玻片和蘸到的第一和第二阱含有CHO生长培养基中的缓冲液冲洗。 </li><li>快速涂抹掉多余的缓冲关闭的背面侧与kimwipe盖玻片并立即放置盖玻片，细胞面朝下，对萃取溶液。 </li><li> 10秒后，拿起镊子盖玻片，立即转移，细胞的一面朝上，到含有4％的多聚甲醛固定缓冲。 </li><li>让固定液中孵育样品在室温下至少15分钟。 </li></ol></li><li>未提取的对照组细胞的制备。要确定总在细胞质中的mRNA的量，这是一个好主意，准备一个未提取的对照样品。 <ol><li>盖玻片上生长的细胞按上述方法制备的（见第2.2节），除此以外，毛地黄皂苷提取步骤（2.2.3）被跳过。 </li><li>固定后，在PBS中萃取所述un-细胞需要透+ 0.1％的TritonX-100处理15分钟，在室温下。 </li></ol></li></ol> 3。 FISH染色<ol><li>设计探针的荧光原位杂交<ol><li>使用预测RNA二级结构的软件，如RNAstructure 5.0 18被折叠的一级序列的mRNA的兴趣。 mRNA的折叠部分的视觉评估，以确定为约50个核苷酸的区域是免费的，主要的二级结构。 </li><li>这个区域的反向互补的合成与Alexa546的荧光基团连接到5&#39;末端探针（购从综合DNA技术）。 </li><li>探针用无RNA酶的水稀释至浓度为100μM，可以储存于-20℃下的股票</li></ol></li><li> FISH探针染色<ol><li>请注意，虽然毛地黄皂苷的提取的细胞不需要进一步的通透性，治疗这些固定的样品与2毫升0.1％的Triton X-100稀释的PBS FISH染色前在室温下的15分钟，有利于减少背景信号。 </li><li>幻灯片，然后用PBS洗涤两次，以除去任何残余的多聚甲醛或Triton X-100。 </li><li> 1X柠檬酸钠缓冲液（150 mM NaCl和15mM柠檬酸钠）中的25％至60％的甲酰胺，然后将细胞洗涤两次。一般而言，对于特定的是50-60个核苷酸长度的FISH探针，使用60％甲酰胺，但的poly（A）mRNA的染色用寡聚（dT）FISH探针，甲酰胺的水平降低至25％。 </li><li>为了制备染色室，一个150毫米的陪替氏培养皿的底部是与水覆盖和石蜡一块漂浮在水面上。菜翻过来的水被除去，从而允许石蜡膜附着的菜的底部。空气泡沫是手动删除kimwipes的。 </li><li>对于每个盖玻片染色，移液管的票面上一个感兴趣的FISH探针的杂交溶液中的100微升下降的教育出版商染色室中。的股票FISH探针在杂交缓冲液（100毫克/毫升的硫酸葡聚糖，1×SSC，1毫克/毫升酵母tRNA，5mM的氧钒核糖核苷复合物，25-60％甲酰胺）与25％至60％甲酰胺的工作浓度的稀释0.2微米。请注意，甲酰胺的量应为所使用的特定的探针进行了优化，并是本SSC洗涤缓冲液中，在相同的浓度（见步骤3.3.3）。 </li><li>盖玻片置于细胞侧面向下到FISH溶液染色室中，并保温5-24小时在37℃下在潮湿的腔室，例如组织培养孵化。 </li></ol></li><li>染色样品的清洗和安装<ol><li>以制备无RNase的洗净区域，放下的带状封口膜在一个水平的平坦的表面上，例如作为一个实验室工作台上。为了确保该封口膜是平的，湿的水平表面，在上面放置的封口膜，并手动删除kimwipes任何气泡。 </li><li>染色室从培养箱中取出，并放置在平坦的水平面上。盖玻片，然后通过移液约500微升每个盖玻片的边缘附近的FISH洗涤缓冲液漂浮。该解决方案将绘制之间的玻片和封口膜通过毛细作用。 </li><li>对于每个盖玻片，1毫升洗涤缓冲液（1×SCC用25-60％甲酰胺，请参阅步骤3.3.3）移液到洗涤区石蜡膜（见步骤3.4.1）。 </li><li>使用镊子，盖玻片中除去从染色室，每个被放置到的洗涤缓冲液的下降，并在室温下孵育5分钟。洗涤过程重复3次。 </li><li>载玻片上，用70％乙醇洗涤，，并干燥kimwipes。 </li><li>对于每个盖玻片上，25μl的安装解决方案（DAPI Fluoromount G，Southern Biotech公司）吸取到幻灯片上。请注意，此溶液中含有4&#39;，6 - 二脒-2 - 苯基吲哚（DAPI），有污渍的DNA的，并允许用于可视化的晶核。 </li><li> USI纳克的钳子和kimwipes，盖玻片的背面的被干燥并吸干过量的FISH洗涤缓冲液不干燥的情况下样品。每个细胞，然后放置盖玻片的一面朝下，到下降的安装解决方案。 </li><li>安装的样本，然后标记，可以储存于4℃。 </li></ol></li></ol> 4。成像和定量<ol><li>成像<ol><li>落射荧光显微镜用FISH染色后的细胞的图像。未转染细胞转染的细胞可以分化明亮的荧光信号，在适当的通道。理想的情况下，得到的各字段也将包含被用来确定用于定量的背景荧光的未转染细胞。 </li><li>对于每个字段的鱼和DAPI通道的图像被收购。此外，如果将细胞染色与蛋白质标记，免疫荧光通道图像还收购。请注意，毛地黄皂苷的提取也可以用来可视化ER结合核糖体和蛋白质11。 </li><li>要确保的字段之间的荧光强度，可以进行比较，每个实验组，对每个通道的曝光时间应保持不变。 </li><li>再次，以确保不同的盖玻片上的细胞的荧光强度之间可以进行比较，应尽量减少每天的日常变化的FISH信号衰减或epiluminescence强度的成像盖玻片在一个单一的会议。 </li></ol></li><li>量化。要量化，本地化的ER的mRNA的量，我们使用Nikon NIS元的软件。然而，其他如图像分析软件ImageJ的都可以使用。 <ol><li>使用的Nikon NIS元的图像分析工具，小区外围的核心概括为单独的区域利益（投资回报率）。 </li><li>对于每个感兴趣区域，荧光强度（I T为总细胞强度，I N为核强度）和区域（A T A N）记录和导出到Excel电子表格中。 </li><li>对于每一个图像，背景荧光强度（I B）是通过记录的非转染的细胞中的强度来确定。如果正在分析的poly（A）mRNA水平，无细胞的区域被选择。 </li><li> ER（在提取的细胞）的总量或细胞质（在未萃取的细胞）的荧光是通过以下公式计算: [A T] [I T - I B] - [A N] [I N - I B] </li><li>核染色强度也可被计算，并用作各种治疗之间的内部控制。由于核的毛地黄皂苷治疗17或核糖体破坏11不受影响，核mRNA的量应保持不变，每个这些处理之间。要计算核荧光使用下面的公式: [A N] [I N - IB] </li><li> ％ER-针对性的细胞质中的mRNA可以计算出的平均ER荧光的30-40摘录细胞（见4.2.4）除以平均30-40未提取的细胞（再次见4.2.4细胞质荧光） 。这是至关重要的，所提取的和未萃取的细胞制备，染色和并行成像。 </li></ol></li></ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAstructure:+software+for+RNA+secondary+structure+prediction+and+analysis.">RNAstructure: software for RNA secondary structure prediction and analysis.</a> BMC Bioinformatics. 11, 129 (2010).</li><li>Lcuyer, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+mRNA+localization+reveals+a+prominent+role+in+organizing+cellular+architecture+and+function.">Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.</a> Cell. 131, 174-187 (2007).</li><li>Lcuyer, E., Yoshida, H., Krause, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+implications+of+mRNA+localization+pathways+in+cellular+organization.">Global implications of mRNA localization pathways in cellular organization.</a> Curr. Opin. Cell Biol. 21, 409-415 (2009).</li><li>Gueroussov, S., Tarnawsky, S. P., Cui, X. A., Mahadevan, K., Palazzo, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mRNA+nuclear+export+kinetics+in+mammalian+cells+by+microinjection.">Analysis of mRNA nuclear export kinetics in mammalian cells by microinjection.</a> J Vis Exp. 46, e2387 (2010).</li></ol>

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不同的亚细胞位点的基因定位，通过转录本mRNA的定位蛋白的相互作用，是一种普遍现象的正确排序的蛋白质，以他们的最终目的地，微调基因表达的亚细胞地方规定区19,20。 使用此处所描述的测定，我们重新编码分泌蛋白的mRNA的是否可以保持在ER中的或翻译的核糖体的存在或不存在的问题的。事实上，我们发现，这些基因的一个子集，有这样的活动11。例如?...

在真核生物中，mRNA编码分泌蛋白和膜蛋白可以有针对性地合作翻译ER信号识别颗粒1,2和可维持的ER通过直接翻译核糖体和translocons在3,4之间的相互作用。然而，无论是基因ER独立的核糖体或翻译时，可以有针对性和维护不清楚，直到最近。以往的研究试图解决翻译是否有独立的表达与ER使用细胞分离技术。因为要求苛刻的化学条件，以解除核糖体ER衍生的囊泡，它可能会破坏潜在的微妙的表达ER协会，这些研究没有定论，提供的证据5-8，对9,10锚定的mRNA的核糖体独立的ER 。 为了避免这些问题，我们已经开发出一种原山坳隔离和图像雌激素受体结合的基因。此过程涉及到一个温和的提取处理，从而有效地消除所有（包括非雌激素受体结合的基因）的细胞的细胞质内容，同时保留内质网的形态和其相关的分子。使用这个协议中，我们已经证明，一个子集的mRNAs的目标，然后保持在ER的核糖体或翻译11独立。

visualization of endoplasmic reticulum localized mrnas in mammalian cells

在真核生物中，大多数的信使RNA（mRNA），编码分泌蛋白和膜蛋白定位于内质网（ER）的表面。然而，这些mRNA的可视化可以是具有挑战性。这是特别真实的mRNA时，只有一小部分是内质网相关和非锁定目标（ 即 "免费"）成绩单阻碍该细胞器，它们的分布。为了监测ER-相关的mRNA，我们已经开发了一种方法，在该方法中，细胞被视为一个毛地黄皂苷萃取溶液，选择性permeabilizes质膜与短曝光，从而除去细胞质内容，同时保持该ER的完整性。再加上荧光原位杂交（FISH）此方法时，人们可以清楚地在荧光显微镜下观察雌激素受体 ​​结合的基因。使用该协议ER关联的程度，无论是散装的poly（A）成绩单或特定的基因可以评估甚至是量化的。在这个过程中，人们可以使用该测定调查的mRNA-雌激素受体的相互作用的性质。

要确定的百分比是ER-相关的mRNA，COS-7细胞的转染质粒中的胎盘碱性磷酸酶（ALPP）或细胞色素P450-8B1（CYP8B1）要么用毛地黄皂苷，然后固定的，或直接固定（参见图1，比较"未萃取Ctrl"键"萃取Ctrl"键）。在两个样品中和ER-相关的mRNA表达的分数被计算出来（ 图2）进行定量的非核荧光。我们的数据清楚地表明，对于两个ALPP和CYP8B1，约60％的细胞质mRNA?...

Watch this Scientific Journal Video about Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells at JoVE.com

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

在这里，我们描述了一种方法，可视化在哺乳动物组织培养细胞的内质网相关的mRNA。这项技术涉及的质膜的选择性通透性毛地黄皂苷以除去细胞质随后通过荧光的内容<em在原位</em杂交技术检测无论是散装的poly（A）mRNA的转录。

在哺乳动物细胞中的内质网定位的mRNA的可视化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

visualization-endoplasmic-reticulum-localized-mrnas-mammalian

Research

JoVE Journal

Biology

14.3K 次观看.  University of Toronto. 在这里，我们描述了一种方法，可视化在哺乳动物组织培养细胞的内质网相关的mRNA。这项技术涉及的质膜的选择性通透性毛地黄皂苷以除去细胞质随后通过荧光的内容

视频: Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing和传代的哺乳动物细胞

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

科研

生物学

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

原位贴壁和非贴壁的哺乳动物细胞的亚细胞分馏

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

在哺乳动物细胞通过显微注射mRNA的核出口动力学分析

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes&#8217; association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions&#160;in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

在培养的哺乳动物细胞大规模并行报告基因分析

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

成像本地CA<sup&gt; 2+</sup&gt;在培养的哺乳动物细胞信号

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

在哺乳动物细胞中的内质网定位的mRNA的可视化

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