1。端粒长度协议19 <ol><li> DNA的提取</li></ol>大多数DNA的提取方法也可使用。我们的实验室更喜欢：Qiagen公司都可套件（＃69506）血源。根据源DNA，如口腔或其他来源的不同，不同的隔离方法也可使用。 <ol start="2"><li>底漆： </li></ol>稀释所有的引物在PCR级水100pmoles/μl股票的浓度，并储存在-20℃直到需要。工作股引物作出了新的，并存储在20℃很短的一段时间（几个月）。奥卡拉汉等人所描述的标准引物用于端粒和β-珠蛋白，对SCG，20 。 引物序列： 端粒1：5&#39;-CGGTTTGTTTGGGTTTGGGTTTGGGTT TGGGTTTGGGTT-3&#39; 端粒2：5&#39;-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTT ACCCT-3&#39; SCG 1：5&#39;-GCTTCTGACACAACTGTGTTCAC的TAGC-3&#39; SCG 2：5&#39;-CACCAACTTCATCCACGTTC的ACC-3&#39; 注意：引物稀释至工作浓度为10pmoles/μl前加入到反应组合。 <ol start="3"><li>连续稀释的基因组DNA： </li></ol>稀释的基因组DNA序列，端粒和单拷贝基因（SCG）创建生成一个标准的CT检测值曲线。所用的基因组DNA的提取从淋巴细胞从血液样本。在这些稀释液所建立的DNA与PCR级H 2 O稀释至各浓度的一个因素为1.68，得到两组使用相同的基因组DNA的系列稀释液。基于试验和错误的起始浓度都得到了优化，并且可能需要使用不同的试剂，仪器，来自其他物种的DNA，细胞类型和SCG使用时，必须调整。 注意：使用的基因组DNA的序列稀释是由轻轻地混合，并等待至少15分钟至一小时befo重新下稀释的DNA。每个稀释需要的时间分解的基因组DNA。对于长期储存，分装系列稀释成PCR条储存在-80°C每个PCR条被解冻，并使用一次，以避免可能破坏DNA的冻融循环。 <ol start="4"><li> RT-PCR反应设置</li></ol>仪器：Roche480光循环仪材料：罗氏光循环仪SYBR绿色测试的DNA样品稀释至终浓度为10纳克/微升。我们测试浓度使用的NanoDrop或量子比特。我们进一步稀释的DNA10ng/μl最终5ng/μl的的。我们不会再次测试浓度。 反应部分组成： <table border="1"><tbody><tr><td fo:width="1.5in"> TELO </td><td fo:width="1.5in"> 1X </td><td fo:width=".25in" rowspan="6"></td><td fo:width="1.5in"> SCG </td><td fo:width="1.5in"> 1X </td></tr><tr><td> H 2 0 &lt;/ TD&gt; <td> 6微升</td><td> H20 </td><td> 6微升</td></tr><tr><td> TELO 1 </td><td> 0.2微升</td><td> SCG 1 </td><td> 0.6微升</td></tr><tr><td> TELO 2 </td><td> 1.8微升</td><td> SCG 2 </td><td> 1.4微升</td></tr><tr><td> RXN组合</td><td> 10微升</td><td> RXN组合</td><td> 10微升</td></tr><tr><td> DNA或串行稀释</td><td> 2微升</td><td> DNA或串行稀释</td><td> 2微升</td></tr></tbody></table> 注：预混无DNA和用过量5％至10％。 双方TELO和SCG一起运行的同一个盘子，使用下面的程序。要精确测试的，至少有三个重复，每个样品，应运行和控制。使用的引物中的变化可能会影响结果。我们的协议已经优化的Roche480光循环仪和我们的引物如下工作。 在itial变性 10分钟变性95°C 循环 95°C 10秒保持 60℃5秒保持 72℃11秒保持和单次采集 根据需要，重复45〜55次 2。端粒酶定量检测（QTD）协议（QTD套件的基础上，通过联合生物科技有限公司货号MT3012） 提取物的制备<ol><li>沉淀细胞或组织。 </li><li>与1X PBS洗一次。 </li><li> Repellet和删除1X PBS * </li><li>细胞沉淀重悬在200μl的1×裂解缓冲液为每10 5 -10 6个细胞，或40-100毫克组织。 </li><li>在冰上孵育30分钟。 </li><li>降速样品在4℃下以12,000×g离心30分钟</li><li> 160微升上层清液转移到新的管中，测定蛋白质浓度。 </li><li>分装和速冻剩余的干冰/乙醇提取物对，储存于-80°C。 </li></ol> *在t他的条件，冷冻细胞或组织中的端粒酶是稳定至少一年。当解冻使用，立即悬浮细胞在1×裂解液。 2.1。含量控制热灭活控制：对于每一个样本，热处理提取物端粒酶活性检测前10分钟在85°C孵育一个额外的控制。 TSR控制模板的标准曲线：执行实时定量PCR的TSR，寡核苷酸与端粒引物包含在套件中生成标准曲线的相似序列使用稀释。 <ol><li>准备1:5系列稀释液的库存TSR浓度（0.5 amoles个/μl），用裂解缓冲液</li><li>执行使用1微升每个TSR稀释，包括股票浓度的的端粒酶检测标准检测。可以被存储在这些稀释液，在4℃下至少2周。 </li></ol> 2.2。端粒酶活性测定使用实时PCR QTD <ol><li>对于每个样本中，添加下面的PCR管或薄壁PCR板（总体积25.0微升）： <ul><li> QTD预混料2×12.5微升</li><li> 1.0微升的细胞或组织提取物</li><li> 11.5μl的PCR合格水</li></ul></li><li>调匀</li><li>程序的实时荧光定量PCR检测系统，按照下面的程序： - 25°C为20分钟（端粒酶反应） - 95℃下和10分钟（PCR初始活化步骤） 35-40周期： - 95℃下30秒（变性） - 60℃下进行30秒（退火） - 72°C，持续30秒（扩展名） </li><li> PCR管放置在热循环仪，并开始循环程序。 </li><li>收集阈值周期或周期完成后的C T值。阈值循环周期ΔRn值有统计学意义的增加是首次发现。 </li></ol> 2.3。数据分析<ol><li> GENERATEA标准曲线使用C T读数和端粒酶活性进行比较。 </li></ol>

没有利益冲突的声明。

端粒长度提供了一个独特的学习压力和老化细胞的细胞标记和衰老的机制提供见解。由于其老化的作用首次被提出，众多的研究已经完成，有关端粒长度的年龄，长寿，与年龄有关的疾病，癌症和压力。几十年来，端粒长度测量的金标准是末端限制性片段分析Southern杂交。然而，最近一个负担得起的，快速和容易执行由Cawthon方法已经演变而来的。使用实时定量PCR，Cawthon提供了一个有用的替代测量...

端粒DNA重复六聚体（TTAGGG）序列，发现在染色体的末端。在每个细胞的复制，这些染色体末端缩短。如果它们变得太短，可以进行染色体端粒末端融合，异常重组和降解。因此，足够维持端粒长度染色体稳定性和细胞保护38中起着重要的作用。基因附近发现染色体的末端端粒长度的保养也是至关重要的，因为DNA复制不能继续1-2染色体的尽头。因此，防止端粒缩短，可以提高电池的稳定性。 许多研究已证实，端粒长度与一个有机体的长寿和疾病状态，如3-4在癌症，糖尿病和心血管疾病的6,7。此外，端粒缩短过度紧张或已与不健康的生活方式，可能是通过促 ​​进细胞过早衰老和死亡9。另一方面，一些研究发现，端粒长度与寿命和年龄有关的疾病39，40，41，有没有显着区别。 端粒缩短的保护细胞的先天机制之一是通过激活其自身端粒酶逆转录酶（TERT）。这种酶和其亚单位，端粒酶RNA（TERC），非编码RNA作为模板使用的端粒添加端粒重复染色体的端部10。虽然端粒酶活性缺席参与维持端粒长度的几种类型的细胞和其他机制，增加端粒酶活性与端粒长度的增加。端粒酶激活已建立细胞损伤和强调，避免过早衰老和死亡的机制之一。例如，较长的特表现在德系犹太人的人口与例外寿命11 lomeres，TERC基因的突变或叔已被证明有助于致命的疾病12，和表观遗传调控端粒酶已被证明有效果与年龄有关的疾病13。自从其发现，端粒长度已经提出在各种动物模型以及人类健康状况的生物标志物，但这些早期的研究采用的方法是难以广泛复制，因为繁琐，漫长而昂贵的。在2002年和2009年进一步调整，Cawthon提出并实现了一个新的，准确，快速，简单的基于PCR的协议，以评估进一步探讨端粒长度和细胞生物学的各个方面，它的作用在衰老和疾病19。 选择正确的一项研究端粒测量方法是必不可少的。目前，有多种方法，用于测量端粒长度，每个都有其自己优点和缺点。传统上，端粒长度的测量采用Southern blot分析末端限制性片段（成绩单），其中包括：一。不切端粒重复序列，以获得成绩单，B的限制性内切酶消化的DNA。这些末端限制性片段的Southern印迹法是通过用端粒探针14，37确定的平均TRF长度。尽管此方法是高度精确的，一个小的变动系数，是直接的措施，可以是有利的，用于测量长度分布，TRF分析是昂贵的，劳动密集型的，至少需要3微克DNA。此方法也是不敏感的短的端粒长度和决心，可以混淆的端粒DNA，它可以检测探头，由于（TTAGGG）N像序列含有15，42。 另一种高度准确的方法测量端粒长度的端粒伸长长度分析（STELA），以单Ë分子PCR为基础的方法，只需要少量的DNA。在该方法中，引物被认识到的富含G的悬在染色体末端的一条染色体上，放大一个特定的染色体的端粒要绑定到一个独特的亚端粒序列。然后，将得到的扩增是可视化的Southern印迹法。此方法的优点是高度精确的，能够检测潜在的离群值端粒16。不幸的是，因为并不是所有的染色体两端有富G和可用的端粒序列，端粒长度只能在特定的染色体上测量，这些测量可能无法代表所有端 ​​粒的长度在小区15。 已经实行的另一种方法是使用肽核酸（PNA）探针检测端粒长度。 原位杂交定量花期 （Q-FISH）技术使我们能够想象的端粒在中期，ST癌宁端粒与PNA探针在它们的大小比例，允许特定染色体的端粒之间的比较。虽然这种方法也可以用于细胞间期，在这里针对不同的染色体的端粒长度不能被检测到，它引入了一个在衰老或不常除以单元17测量时，端粒长度的限制。流鱼，而不是使用流式细胞仪，是目前最敏感的方法，在临床上用于测量血细胞的端粒长度，但需要高度熟练的技术人员18。 目前，在我们的实验室中测量平均端粒长度的标准方法充分利用的精度，灵敏度，简便的定量实时PCR。此方法所开发的新型引物，避免合成的引物二聚体衍生的产品，否则会被作为测量端粒长度尽可能由于端粒的重复性质TANDARD测定。对于这个实验中，端粒长度的测量T / S比，端粒重复序列拷贝数单拷贝基因数目表示。因为有一个直接的端粒长度和标记的端粒引物结合的DNA的PCR过程的开始阶段的数量之间的比例关系，在T / S比率成正比的端粒长度。 T / S比值衡量比较CT的差异，在该样品的循环数累计花期越过一个门槛就是几基线花期个标准差以上，端粒引物与样品之间的和SCG引物19。这种方法已经被批评为间接测量端粒长度，这可能会导致不准确的测量，例如，在染色体重复或拷贝数变异15的情况下。此外，研究之间的比较往往是二已发展到测量绝对端粒长度20 fficult，但标准低聚物。此方法进一步改进通过Cawthon，使用单色多重qPCR实验。在该试验中，在PCR的前几个周期，在较低温度下运行，以避免引物二聚体的结合，并的端粒和控制基因进行了分析，在相同的PCR管中以进一步避免错误。与端粒长度的末端限制性片段的Southern印迹分析测定，将所得的端粒长度测量很强的相关性，并具有更高的精度15，19，36。此刻，实时定量RT-PCR是唯一方便的方法，可用于大样本测试，只需要少量的DNA进行。这种方法的一个重要部分是全面的质量控制措施，这样，当它处理得当，这种方法可以提供有价值的比较信息端粒长度。此外，这种方法已被改编为超越白细胞测量在各种不同的组织42的端粒长度。 此外，为了进一步了解背后的生物学机制维持端粒长度和两种相反的效果（ 即端粒缩短和伸长率在复制过程中的端粒酶）之间的相互作用，我们伴随着额外的检测，测量端粒酶活性与端粒长度的方法。 为了这个目的，我们使用了端粒重复序列扩增， 在体外试验。简单地说，溶解，保护，合成的酶活性的细胞将端粒重复序列的寡核苷酸的基板使用端粒酶，并用PCR扩增的存在下的SYBR Green的产品。结果，然后进行分析比较样品和控制CT阈值。由于端粒酶是一种对热敏感的酶，一个额外的热处理控制每个样品21一 ​​起运行。 <p class=的“jove_content”&gt;测量端粒长度的同时，可以提供有价值的洞察衰老和疾病的病理生理中端粒长度可能发挥的作用，端粒长度是不是一个静态的财产和更多的信息，以便了解端粒功能。端粒酶活性的研究可以添加信息端粒长度的调控机制。例如，控制端粒酶活化可以维持端粒长度和细胞增殖，但端粒酶的不受控制的激活可以导致癌症。这两个价格低廉，直远期相结合的方法，不仅为我们提供有力的证据，对端粒长度和细胞稳定性之间的关系，同时也进一步了解端粒缩短和恢复的可能机制。有了这些方法，我们希望进一步阐明细胞的老化和细胞增殖的各种状态，最终目标是更好地理解中央机制及ms的细胞生物学。

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Quantitative Telomerase Detection </td>
 <td>Allied Biotech </td>
 <td>MT3012 </td>
 </tr>
 <tr>
 <td>Qiagen DNeasy Kit</td>
 <td>Qiagen</td>
 <td>69506</td>
 </tr>
 <tr>
 <td>LightCycler 480 SYBR Green I Master, Ready-to-use hot start reaction mix for SYBR Green I-based real-time PCR using the LightCycler 480 Instrument</td>
 <td>Roche Diagnostics</td>
 <td>4707516</td>
 </tr>
 </tbody>
</table>

telomere length and telomerase activity; a yin and yang of cell senescence

端粒重复DNA序列结束的尖端处的染色体长度和人类中是多种多样的，可以达到15000个碱基对的长度。端粒在每次细胞分裂染色体磨损作为一个生物保护机制。在一定的长度，端粒变得太短，允许复制，这个过程可能导致染色体不稳定或细胞死亡。端粒长度是受两个对立的机制：磨损和断裂伸长率。每个细胞分裂发生磨损。与此相反，伸长率部分调制的端粒酶，这增加了重复序列的染色体的端部。以这种方式，端粒酶可能扭转老化机制，活化细胞活力。这些都是重要的元素，在维持细胞的生命和用于评估细胞的衰老。在这篇稿件中，我们将介绍一个准确的，短的，复杂的和便宜的方法来评估在多个组织的端粒长度s和物种。这种方法需要利用两个关键要素，串联重复端粒序列和定量RT-PCR检测的灵敏度差分被测样品的拷贝数。此外，我们将介绍一个简单的检测，以评估作为一个互补的骨干测试端粒长度的端粒酶活性。

端粒长度定量RT-PCR分析的一个例子是图1中所示。在左上角的面板中，在红色和绿色标记的样品代表的位置上的96 - 孔板的受试者。在右上方的面板，扩增曲线证明。每个主题是由两个检测（端粒，单拷贝基因），这是一式三份进行测试。由于选定的个人的两个实验中产生的不同的DNA的拷贝数，花期检测的周期数，直到达到其指数曲线之间的不同检测。在较高的拷贝数管（ 即端...

Watch this Scientific Journal Video about Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence at JoVE.com

Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence

描述一个准确的，短的，复杂的和廉价的方法，使用定量RT-PCR在多个组织和物种评估端粒长度。此外，我们将介绍一个简单的检测，以评估作为一个互补的骨干测试端粒长度的端粒酶活性。

端粒长度和端粒酶活性;阴阳细胞衰老

Telomeres  are repeating DNA sequences at the tip ends of the chromosomes that are diverse  in length and in humans can reach a length of 15,000 base ...

telomere-length-telomerase-activity-yin-yang-cell

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Molecular Biology

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Unit 1

DNA Replication

SCIENTISTS IN ACTION

46.3K 次观看.  Albert Einstein College of Medicine. 描述一个准确的，短的，复杂的和廉价的方法，使用定量RT-PCR在多个组织和物种评估端粒长度。此外，我们将介绍一个简单的检测，以评估作为一个互补的骨干测试端粒长度的端粒酶活性。

视频: Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence

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 ...

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生物学

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)&#x2013;dgn fusion protein (GFP&#x2013;dgn, unstable) and a variant carrying a frameshift mutation (GFP&#x2013;dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP&#x2013;dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

（DGN）一Degron不稳定的绿色荧光蛋白（GFP）的报告蛋白在活细胞内泛素 - 蛋白酶体活性的监测

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

ECIS的/出租车细胞趋化测量

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

在体外细胞迁移和侵袭实验

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

样机小型化流式细胞仪及配套的混合微流控技术降低重力环境硬件演示

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

关联特定基因的DNA甲基化与表达的变化和星形胶质细胞的转录活性<em&gt; KCNJ10</em&gt;（Kir4.1）

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

单探头的应用：根据环境条件质谱成像和单细胞分析

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology of the fish branchial skeleton is matched to a species' diet. Threespine stickleback fish (Gasterosteus aculeatus) have emerged as a model system to study the genetic and developmental basis of evolved differences in a variety of traits. Marine populations of sticklebacks have repeatedly colonized countless new freshwater lakes and creeks. Adaptation to the new diet in these freshwater environments likely underlies a series of craniofacial changes that have evolved repeatedly in independently derived freshwater populations. These include three major patterning changes to the branchial skeleton: reductions in the number and length of gill raker bones, increases in pharyngeal tooth number, and increased branchial bone lengths. Here we describe a detailed protocol to dissect and flat-mount the internal branchial skeleton in threespine stickleback fish. Dissection of the entire three-dimensional branchial skeleton and mounting it flat into a largely two-dimensional prep allows for the easy visualization and quantification of branchial skeleton morphology. This dissection method is inexpensive, fast, relatively easy, and applicable to a wide variety of fish species. In sticklebacks, this efficient method allows the quantification of skeletal morphology in genetic crosses to map genomic regions controlling craniofacial patterning.

The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species.

解剖和三刺鳃骨架的平面安装

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology ...

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

技术诱导和量化细胞衰老

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...