收养的DTH诱导 请看到的Jove文章 ： <a href="/index/Details.stp?ID=325">收养大鼠迟发型超敏反应的感应和</a>监测 恭Beeton，乔治K。Chandy 生理学和生物物理学系，美国加州大学欧文分校 可视化实验研究，第8期在这里，我们使用1:1的比例与未​​标记的抗原的抗原（卵清蛋白）结合德州红，让吞噬抗原提呈细胞的可视化修改这一协议。 耳组织收获<ol><li>安乐死在批准的动物协议中列出的程序所需的时间点，大鼠。在这里，我们使用过量的麻醉，异氟醚。这是在位于通风良好的引擎盖内的密闭容器中。确保动物是要么打开胸腔，切心斩首的死。 </li><li>使用大剪刀移除一个单一的切割，尽可能接近动物体内（图1）耳。 <img src="/files/ftp_upload/old/907_2008_8_13_0/Figure_1.jpg" alt="Figure_1.jpg" /> 图1。去除鼠耳 </li><li>放置在一个含有冰冷的1X PBS或RPMI - 1640〜10毫升15毫升锥形管耳。置于冰上，直到您准备，以备成像组织的组织样本。 </li><li>图片组织尽快，我们发现，虽然1至2小时在冰上，在组织没有可辨别的影响细胞的运动相比，收获和成像立即组织。 </li></ol>成像的组织准备一，去除表皮和真皮层 *注 ：深真皮层，必须保持完整。如果做得正确，大血管，将保持不变，耳朵软骨不会被暴露。这是一个很难的技术，特别是当有没有炎症控制条件和使用解剖显微镜，它是有帮助的。 <ol><li>将组织从10 cm组织培养皿中的锥形管和媒体。 </li><li>带着手套的双手抱大鼠耳边轻轻耳朝上（图2）耳背侧（远端）的一角。 <img src="/files/ftp_upload/old/907_2008_8_13_1/Figure_2.jpg" alt="Figure_2.jpg" /> 图2。修剪皮毛断耳 </li><li>用你的拇指之上的耳朵，稳定的组织，而使用封闭杜蒙提示＃5剪刀分开真皮深层真皮。另外，如果耳朵边缘的皮肤/真皮深层悬，这可以掌握用镊子轻轻拉分开，从下面的组织（图3）。 <img src="/files/ftp_upload/old/907_2008_8_13_2/Figure_3.jpg" alt="Figure_3.jpg" /> 图3。去除真皮上部 </li><li>一旦小面积的皮肤已经从底层组织分开，重复的分离步骤，或者使用之间的真皮层和镊子剪刀削减轻轻地清除和剥离脱落的皮肤。 </li><li>揭露一个5 x 5毫米或更大的耳组织，从最初的抗原注射网站（图4）至少有3毫米的面积。 <img src="/files/ftp_upload/old/907_2008_8_13_3/Figure_4.jpg" alt="Figure_4.jpg" /> 图4。组织成像暴露 </li></ol>二。保护组织的成像阶段<ol><li>剪下一个稍大比准备组织大小的塑料盖滑。 </li><li>传播3M Vetbond™安全组织跨幻灯片胶薄薄的一层。 </li><li>抓住准备组织（你是不是有可能形象）的角落，从媒体中删除，并组织民建联的镜头纸或许姆底面擦拭，除去多余的介质。 </li><li>触摸胶水覆盖的幻灯片（湿纸巾立即Vetbond治愈）组织的边缘。轻轻地舒展组织扁平，最后触摸幻灯片（图5）与异性结束 <img src="/files/ftp_upload/old/907_2008_8_13_4/Figure_5.jpg" alt="Figure_5.jpg" /> 图5。摩组织盖玻片 </li><li>固化Vetbond™反相组织样本/幻灯片和水库的媒体，如组织和幻灯片在45度角朝下，大约浸泡。倒挂控股的角度组织，有利于暴露组织表面固化，以防止多余的胶水。组织表面上胶块激光（图6）。 <img src="/files/ftp_upload/old/907_2008_8_13_5/Figure_6.jpg" alt="Figure_6.jpg" /> 图6。固化vetbond组织GLUE的媒体浸渍 注意 ：步骤2-5可能需要稍加练习掌握。先试后首次试图组织块丢弃或额外练习。 </li><li>民建联的少量硅脂到幻灯片的底部。 </li><li>放置在灌注室的幻灯片。确保灌注媒体是流动的，37℃，并加入组织前含氧。轻轻向下按幻灯片灌注腔的底部和塑料盖滑之间的硅脂密封边缘。 </li></ol>三。双光子成像<ol><li>使用明场透射光，着眼于组织的顶部。关闭所有的灯。 </li><li>打开激光，光电倍增管所要求的多光子显微镜系统。 </li><li>高度有序的纤维蛋白二次谐波（SHG）（胶原蛋白和肌球蛋白）产生的信号在激发光的波长的一半。我们使用900纳米的激发，导致在450 nm处，它可以可视化使用“蓝色”通道显微镜倍频。倍频一代，是一种非线性的（双光子）的过程中，从而提供了光学切片“双光子激发荧光效果相似。倍频峰效率时，会发生纤维垂直于激光并行运行的纤维，而不会被显示。 </li><li>用大量的细胞中找到一个很好的成像面积，购置面积等，多数细胞在中心，并开始成像设置。注：在DTH反应，T细胞及其他细胞浸润经常发现被集中到了一起，而其他地区都没有浸润细胞 2 。因此，它可能需要一些时间，寻找组织，找到最好的地区形象。 </li><li>检查评估期间的成像速度和极性细胞细胞活力。使用最低的激光功率，提供可接受的图像质量，以确保最小的光损伤。 </li><li>交换需要一个新的组织准备。 </li></ol>

<ol>
	<li>Koo, G. C., 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=Blockade+of+the+voltage-gated+potassium+channel+Kv1.3+inhibits+immune+responses+in+vivo.">Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo.</a> J Immunol. 158, 5120-5128 (1997).</li><li>Gaga, M., Frew, A. J., Varney, V. A., Kay, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eosinophil+activation+and+T+lymphocyte+infiltration+in+allergen-induced+late+phase+skin+reactions+and+classical+delayed-type+hypersensitivity.">Eosinophil activation and T lymphocyte infiltration in allergen-induced late phase skin reactions and classical delayed-type hypersensitivity.</a> J Immunol. 147, 816-822 (1991).</li><li>Flugel, A., Odoardi, F., Nosov, M., Kawakami, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoaggressive+effector+T+cells+in+the+course+of+experimental+autoimmune+encephalomyelitis+visualized+in+the+light+of+two-photon+microscopy.">Autoaggressive effector T cells in the course of experimental autoimmune encephalomyelitis visualized in the light of two-photon microscopy.</a> J Neuroimmunol. 191, 86-97 (2007).</li><li>Kawakami, N., 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=Live+imaging+of+effector+cell+trafficking+and+autoantigen+recognition+within+the+unfolding+autoimmune+encephalomyelitis+lesion.">Live imaging of effector cell trafficking and autoantigen recognition within the unfolding autoimmune encephalomyelitis lesion.</a> J Exp Med. 201, 1805-1814 (2005).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1873 (2002).</li><li>Matheu, M. P. B., A, C. G. a. r. c. i. a., Chi, V., Rangaraju, S., Safrina, O., Monaghan, K., Uemura, M. I., Li, D., Pal, S., Monuki, E., Flugel, A., Pennington, M. W., Parker, I., Chandy, G. K., Calahan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Situ+Imaging+of+Effector/Memory+T+Cells+During+DTH+and+Suppression+by+Kv1.3+Channel+Block.">In Situ Imaging of Effector/Memory T Cells During DTH and Suppression by Kv1.3 Channel Block.</a> Immunity. , (2008).</li></ol>

imaging effector memory t cells in the ear after induction of adoptive dth

迟发型超敏反应（DTH）是主体CCR7的-效应/记忆性T淋巴细胞免疫反应。在这里，我们展示了一种诱导和记录在大鼠耳的DTH反应的进展的方法。这是由双光子成像的CCR7的示范-效应/记忆T细胞应答鼠耳组织编制。 收养的DTH诱导GFP标记的OVA特定CCR7的腹腔注射 -效应/记忆T细胞系（Beeton，C研究的可视化实验，第8期） 。然后让细胞在大鼠平衡，1:1混合的卵子和卵子的共轭得克萨斯红（OVA - TR）的一只耳朵注射用生理盐水（对照耳）和其他允许可视化挑战48小时前居民的抗原提呈细胞。 我们描述了一个有用成像结合胶原纤维的二次谐波产生的可视化过程中的免疫反应，深层的真皮层内的细胞活力的组织准备的方法。耳组织切成5 × 5毫米平方（稍大更好），装到塑料盖使用Vetbond™单，然后安全的使用在成像室硅脂和37氧冒泡的组织培养液灌流° C 。

Watch this Scientific Journal Video about Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH at JoVE.com

Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

在这里，我们证明了诱导和记录一个迟发型超敏反应（DTH）反应在鼠耳进展的方法。这是由鼠耳组织编制的示范效应/记忆性T细胞反应的双光子成像。

成像效应在耳朵上的记忆体T细胞诱导后收养的DTH

Delayed type hypersensitivity (DTH) is an immune reaction in which the main players are CCR7- effector / memory T lymphocytes. Here, we demonstrate a ...

imaging-effector-memory-t-cells-ear-after-induction-adoptive

Nanyang Technological University

Research

JoVE Journal

Biology

10.2K 次观看.  University of California, Irvine (UCI). 在这里，我们证明了诱导和记录一个迟发型超敏反应（DTH）反应在鼠耳进展的方法。这是由鼠耳组织编制的示范效应/记忆性T细胞反应的双光子成像。

视频: Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

Induction and Monitoring of Active Delayed Type Hypersensitivity (DTH) in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin (KLH).
Here we demonstrate how to induce an active DTH reaction in Lewis rats. We will first prepare a water-in-oil emulsion of KLH, our antigen of interest, in complete Freund's adjuvant and inject this emulsion subcutaneously to rats. This will prime the immune system to develop memory T cells directed to KLH. Seven days later we will challenge the rats intradermally on the back with KLH on one side and with ovalbumin, an irrelevant antigen, on the other side. The inflammatory reaction will be visible 16-72 hours later and the red and swollen area will be measured as an indication of DTH severity.

Induction and Monitoring of Active Delayed Type Hypersensitivity DTH in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes. Here we demonstrate how to induce active DTH in Lewis rats and monitor the inflammatory response.

大鼠主动迟发型超敏反应（DTH）的诱导和监控

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

科研

生物学

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

收养大鼠迟发型超敏反应的感应和监测

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

克隆形成含量:贴壁细胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

发展中国家斑马鱼的表皮细胞挤压实时成像

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

使用数字免疫荧光显微镜成像在大鼠软脑膜小动脉雌激素受体α

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

荧光成像与一纳米精度（FIONA）

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

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, ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...