我们想感谢我们的程序和罗德尼McPhail（生物科学系，美国普渡大学）寻求协助拍摄与编辑夹层视频瑞安Maneri（Oystercatcher制作，LLC）。在苏特尔实验室的研究是支持由美国国立卫生研究院（R01 NS049233）和Bindley生物科学中心，在美国普渡大学的资助到DMS

解决方案<ul><li> L15 - ASW的细胞培养液（1L） <ul><li> L15粉1袋</li><li>加入800毫升的H 2 O超纯</li><li>氯化钠400毫米</li><li>硫酸镁4 27毫米</li><li> MgCl 2的 28毫米</li><li> L -谷氨酰胺4毫米</li><li>庆大霉素50微克/毫升</li><li> HEPES 5毫米</li><li>调整pH值7.9 </li><li>添加一滴一滴氯化钙2 9.3毫米（停止，如果沉淀物） </li><li>加入H 2 O超纯水至1 L </li><li>检查渗透压与渗压计（蒸气压力，Wescor＃5520）（950-1000毫摩尔/公斤） </li><li>过滤器采用0.22微米正压过滤装置（过滤器：微孔SVGV010RS） </li><li>在4 ° C存储长达1个月</li></ul></li><li>聚- L -赖氨酸（70-150 KD）200微克/毫升的无菌H 2 O超纯原液保存在-20℃ </li><li> 0.5 M MgCl 2的解决方案</li></ul>第1天：海兔的腹神经节的解剖制备的酶解解决方案<ol><li> Eppendorf管中，称取9-10毫克Dispase酶（沃辛顿，猫：LS02111） </li><li> L15及反潜培养基，添加900μL和100μL无菌H 2 O超纯</li><li>混合并放置在室温（RT） </li></ol>腹神经节的解剖<ol><li>填写50毫升的注射器，用0.5 M MgCl 2的解决方案。 </li><li>坦克从一个动物，轻轻地处理，以避免动物的墨防御反应。 </li><li>在夹层板在其一侧的海兔（聚苯乙烯泡沫板），喙侧的权利，尾椎的一面放置到左边。 </li><li>针插入动物仅次于头部和体腔注入所有MgCl 2的解决方案。 </li><li>轻轻揉搓动物驱散MgCl 2的解决方案，整个体腔，等待几分钟动物完全麻醉。 </li><li>引脚的动物的头部和尾部的夹层板与两针。 </li><li>使用镊子，解除皮肤，通过对头部和尾部的大剪刀的皮肤和肌肉层切开，使上侧的大开放。 </li><li>查找毛刷状卵巢下腹部神经节。使用镊子举行约1厘米在前面的腹神经节的连接神经，降低清扫剪刀钳控股地位的前面和后面的腹神经节神经。 </li><li> Dispase解决方案，在22孵育15-16 h的神经节放入℃使用温度控制水浴。 </li></ol>第2天：海兔袋细胞电镀准备多聚赖氨酸涂层盖玻片<ol><li>在流台，准备3个35毫米培养皿中的神经节清扫工作的美味佳肴，灌装4毫升L15及反潜中等每个。腹神经节从dispase解决方案转移工作15.5小时消化后的菜肴之一。 </li><li>使用钳取酸清洗＃1.5盖玻片（22x22毫米）乙醇中，除去酒精和消毒本生灯燃烧超过他们简要盖玻片。让他们简单地冷却，然后放入培养皿中的盖玻片。 </li><li>准备一个黄色的提示在本生灯（火焰只是简单地避免塑料熔化！）弯曲弯曲的镀尖。 </li><li>准备适量的20微克/毫升的多聚赖氨酸的解决方案，200微克/ 毫升 ，用无菌H 2 O超纯原液稀释。 </li><li>每个盖玻片覆盖500μL的20μg/ ml的多聚赖氨酸和20分钟在室温下孵育。 </li><li>与500μL无菌H 2 Ø超纯每个洗盖玻片每3-5倍。 </li><li>从盖玻片取出的水;填写L15及反潜培养液4毫升每道菜。 </li></ol>离解袋细胞<ol><li>一个夹层显微镜下，削减一半使用的清扫剪刀和以前用95％乙醇（图1 1号线）清洗钳腹神经节。 </li><li>在切袋细胞集群和hemiganglion细胞（图1，2号线）之间的神经节，并切去袋细胞的另一边的剩余的神经扩展（图1，第3行）的一半。一节半时间的工作，留在盘的另一半，而从第一个群集细胞都被镀。 </li><li>利用两个钳（或一个钳和清扫剪刀），释放袋推开袋细胞群周围的结缔组织鞘细胞群。清扫剪刀剪去结缔组织，如果需要的话。小心不要挤在这个过程中太多触摸袋细胞群。 </li><li>当大多数的结缔组织被删除，转移的BAG细胞群集准备工作进入一个新盘的弯曲的黄色提示。要小心，不要让袋细胞群被困在里面的黄色提示或滞留在空气/介质界面。 </li><li>磨碎的黄色的提示，删除单个袋细胞群。吸取集群和针尖。开始与低剪切力，需要增加力量。始终使用最低的力量，需要删除没有过多的脑细胞死亡的神经元。 </li><li>收集了3-5健康袋细胞，并与多聚赖氨酸涂层的盖玻片，他们转移到菜。避免拿起死细胞（黑色/透明的中心）和结缔组织碎片。 </li><li>重复步骤6。 15-25活神经元的每个盖玻片中心举行。如果死细胞或碎片，以及转移，删除不需要的材料，拿起它，或从健康细胞中，它吹。这个过程需要两个集群约为2-3小时。 </li><li>一旦完成，流的长椅上的培养皿保持在室温下至少2小时，让细胞附着（保持流板凳鼓风机关闭，以避免不必要的振动）。 </li><li>在14 ° C过夜，或直至进一步使用，放入孵化器的菜肴。生长锥可能会在4-10小时。 </li></ol><img src="/files/ftp_upload/old/662_2008_2_10_0/image001.jpg" alt="图1。海兔californica腹神经节的示意图。颜色的线条表示削减获得袋细胞簇。" /> 图1。 海兔californica腹神经节的示意图。颜色的线条表示削减获得袋细胞簇。 第3天：海兔袋细胞实时成像室大会的成像室<ol><li>使用包活细胞成像实验细胞之前，低功耗显微镜检查细胞。如果细胞还没有开发的健康生长锥，在室温下供应新鲜培养基和休假1-2个小时。 </li><li>供应新鲜L15及反潜介质板，真空线删除旧媒体（〜2毫升）的一半，并加入2 ml新的L15 - ASW的每培养皿中的介质。重要的是不完全删除所有媒体从盘一次，因为这将消除细胞的盖玻片。 </li><li>袋细胞成像，我们聚集在两个盖玻片和两个塑胶垫片，类似于三明治的成像室。夹心顶部盖玻片被切断短允许容易的培养基和试剂交流。在装配过程中，与袋的贴壁细胞的盖玻片应被淹没在任何时候，以防止其移动细胞L15及反潜培养基。 </li><li>准备室，切断〜＃1盖玻片22x22毫米钻石笔（玻璃切割机）和标尺的右侧2毫米。 </li><li>用1 ml注射器，适用于高真空润滑脂较短盖玻片两侧，并连接每边的塑胶垫片。应用真空润滑脂两个垫片。 </li><li>反转盖玻片和间隔大会，并将其放置到培养皿包含一个玻璃罩上碟袋细胞。对齐并轻轻向下按压顶部底部的盖玻片盖玻片坚持两个Q - TIPS反向。 </li><li>使用镊子，轻轻夹心大会的培养皿中无泄漏出来的媒介。对齐两个盖玻片，如果需要的话，一个Kimwipe擦去多余的介质。放置到显微镜幻灯片持有人的底部夹层和附加使用剪辑的护理外壳。 </li><li>清洁三明治的顶面中心与Q - TIPS擦了擦玻璃清洁剂（旌宇玻璃清洁剂）。第二，干棉条的玻璃清洁，以避免连败线快速干燥。底部的盖玻片相同。免费清洁盖玻片连胜线是重要的高品质的影像。 </li></ol>袋细胞在显微镜由于海兔袋细胞神经元文化的密度低，这是最好的，如果您的显微镜设置阶段的定位功能，促进细胞之间的的切换。袋细胞可以很容易地保存在舞台上的几个小时在成像实验在室温下。定期交换的媒介，以避免蒸发。

<ol>
	<li>Forscher, P., Kaczmarek, L. K., Buchanan, J. A., Stephen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+induces+changes+in+distribution+and+transport+of+organelles+within+growth+cones+of+Aplysia+bag+cell+neurons.">Cyclic AMP induces changes in distribution and transport of organelles within growth cones of Aplysia bag cell neurons.</a> J. Neurosci. 7, 3600-3611 (1987).</li><li>Suter, D. M., Errante, L. D., Belotserkovsky, V., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ig+superfamily+cell+adhesion+molecule,+apCAM,+mediates+growth+cone+steering+by+substrate-cytoskeletal+coupling.">The Ig superfamily cell adhesion molecule, apCAM, mediates growth cone steering by substrate-cytoskeletal coupling.</a> J. Cell. Biol. 141, 227-240 (1998).</li><li>Schaefer, A. W., Kabir, N., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filopodia+and+actin+arcs+guide+the+assembly+and+transport+of+two+populations+of+microtubules+with+unique+dynamic+parameters+in+neuronal+growth+cones.">Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones.</a> J. Cell. Biol. 158, 139-152 (2002).</li><li>Suter, D. M., Schaefer, A. W., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+dynamics+are+necessary+for+SRC+family+kinase-dependent+growth+cone+steering.">Microtubule dynamics are necessary for SRC family kinase-dependent growth cone steering.</a> Curr. Biol. 14, 1194-1199 (2004).</li></ol>

海兔袋细胞神经元提供了很少的非神经元细胞的无血清神经元细胞培养系统。这些神经元生长锥形成非常大的，适合解决一些重要的细胞生物学问题。袋细胞神经元可以很容易地被操纵，并在室温下成像几个小时。使用荧光斑点显微镜（密克罗尼西亚），可以定量分析的F -肌动蛋白和微管聚合和易位动态的各种参数。这些成像工具，与最近发布的海兔的基因组信息，以及改进的表达技术，使这些神经元的一个功?...

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		<th>Material Name</th>
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		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>#1 glass coverslips 22x22 mm</td>
<td>Tool</td>
<td>VWR</td>
<td>48366067</td>
<td>&nbsp;</td>
<tr>
<td>#1.5 glass coverslips 22x22 mm</td>
<td>Tool</td>
<td>Corning</td>
<td>2870-22</td>
<td>&nbsp;</td>
<tr>
<td>35 mm Petri dishes</td>
<td>Tool</td>
<td>Falcon</td>
<td>353001</td>
<td>&nbsp;</td>
<tr>
<td>Filters for medium filtration</td>
<td>Tool</td>
<td>Millipore</td>
<td>SVGV010RS</td>
<td>&nbsp;</td>
<tr>
<td>High vacuum grease</td>
<td>Tool</td>
<td>Dow Corning</td>
<td>1597418</td>
<td>&nbsp;</td>
<tr>
<td>Osmometer</td>
<td>Tool</td>
<td>Wescor</td>
<td>5520</td>
<td>&nbsp;</td>
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<td>Plastic shims</td>
<td>Tool</td>
<td>Small Parts Inc </td>
<td>SHSP-200</td>
<td>&nbsp;</td>
<tr>
<td>Calcium chloride</td>
<td>Reagent</td>
<td>Fisher</td>
<td>C79-500</td>
<td>&nbsp;</td>
<tr>
<td>Gentamicin</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15750-060</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H4030</td>
<td>&nbsp;</td>
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<td>L15 medium</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>41300-039</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>G8540</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium chloride</td>
<td>Reagent</td>
<td>Mallinckrodt</td>
<td>5958-04</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium sulfate</td>
<td>Reagent</td>
<td>Mallinckrodt</td>
<td>6070-12</td>
<td>&nbsp;</td>
<tr>
<td>Poly-L-lysine (70-150 kD) </td>
<td>Reagent</td>
<td>Sigma</td>
<td>P6282</td>
<td>&nbsp;</td>
<tr>
<td>Sodium chloride</td>
<td>Reagent</td>
<td>Mallinckrodt </td>
<td>7581</td>
<td>&nbsp;</td>
<tr>
<td>Neutral Protease (Dispase)</td>
<td>Reagent</td>
<td>Worthington</td>
<td>LS02111</td>
<td>&nbsp;</td>		</tbody>
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neuronal cell cultures from aplysia for high-resolution imaging of growth cones

神经生长锥的轴突，可以检测到环境中的指导线索，并传导到对相应的靶细胞的定向运动信息提示的高度能动结构。要充分了解信息，指导如何从细胞表面传输到底层的动态骨架网络，需要适合高时空分辨率的成像在活细胞的蛋白质动力学模型系统。典型的脊椎动物生长锥是太小了定量分析的F -肌动蛋白和微管动力学。从海兔海兔californica的神经元是脊椎动物的神经元的5-10倍，比大，可以很容易地在室温下保持的，是非常强大的细胞显微和生物物理测量。他们的生长锥有非常明确的细胞质地区和一个很好的描述的骨架系统。显微注射神经细胞机构可以与各种研究生长锥的运动和指导的探头。在本议定书中，我们展示了一个过程，解剖腹腔神经节，培养袋细胞神经元，并设立一个生长锥的活细胞成像的成像室。

Watch this Scientific Journal Video about Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones at JoVE.com

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

海兔californica神经元发展大文化的生长锥，生长锥的运动和指导的高分辨率成像的优秀。在这里，我们提出了一种海兔袋细胞神经元的剥离和电镀以及设立一个活细胞成像室的协议。

从海兔的神经细胞培养的高分辨率成像生长锥

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

neuronal-cell-cultures-from-aplysia-for-high-resolution-imaging

Research

JoVE Journal

Biology

11.3K 次观看.  Purdue University. 海兔californica神经元发展大文化的生长锥，生长锥的运动和指导的高分辨率成像的优秀。在这里，我们提出了一种海兔袋细胞神经元的剥离和电镀以及设立一个活细胞成像室的协议。

视频: Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

从晚期果蝇蛹的大脑原发性神经文化

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

科研

生物学

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

从Midgastrula舞台果蝇胚胎神经文化的制备

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization 3. Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro 3. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. 
Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later andexcess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20&deg;C.

We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.

准备从大鼠脾细胞T细胞生长因子

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

高分辨率视频跟踪移动的，在成年果蝇。

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

制备海兔感觉运动神经元细胞培养

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

原代细胞培养从果蝇原肠胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

的分散Presomitic中胚层细胞培养的斑马鱼分割时钟在单细胞成像代

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

在啮齿类动物呼吸道上皮纤毛运动功能的高分辨率成像的体外方法

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

使用plusTipTracker软件测量微管动力学<em&gt;非洲爪蟾</em&gt;生长锥

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...