新西兰想感谢她的实验室成员Subhas比斯瓦斯，安德鲁Sproul，和瑞安威利特培训，在解剖和收获交感神经元。由NIH - NINDS赠款支持。

1。解剖的筹备工作： <ol><li>选择适当的培养皿（10厘米或6孔板或24孔板等）取决于您的实验性质，和大衣与鼠尾胶原24小时前清扫。准备鼠尾胶原蛋白，并用它来大衣培养皿的方法已在别处（1）。 </li><li>准备0.25％胰蛋白酶溶液中的磷酸缓冲液（PBS）（无EDTA）和过滤消毒。准备1毫升分装和存储-20℃ </li><li>准备一个解决方案包含在蒸馏水/去离子H 2 O 2.5毫米尿苷和5 - FDU过滤消毒，并准备50μL分装。贮存于-20℃ </li><li>准备完全培养基：10％热灭活马血清（热灭活，在56 º C水浴孵育30分钟）和5％胎牛血清的RPMI 1640培养液。 </li><li>准备最终中等：用1％热灭活马血清（100ηg/ml终浓度NGF）+青霉素/链霉素（1 / 250）+ 1 / 250 uridine/5-FDU解决方案的RPMI 1640培养基（终浓度为10μM） 。注意：这个媒介应新鲜，每次使用前。实验在24孔板格式，将需要准备12毫升这种媒介。建议你准备一点点额外的。 NGF可以从各种商业来源（见表）在本议定书年底购买。 </li><li>设备：一是需要在解剖显微镜和光源。重点提示一个双鹅颈纤维光纤照明是首选。此外，将需要清洗和消毒（高压灭菌或用70％乙醇浸泡至少20分钟）以下的清扫工具：两对微解剖不锈钢镊子，和一双解剖剪刀（不锈钢从Roboz）。也需要两个无菌注射器的针头（26 gague × 1 ½英寸或类似）。有必要成立一个夹层板，这是一个3“X3”一块发泡胶包裹在铝箔和Kimwipe覆盖。喷雾用70％乙醇，以净化板。最后，准备一个火抛光的玻璃牧场吸管。以火抛光的玻璃吸管，并保持在几秒钟的火焰提示，同时旋转。检查后，提示会看起来更加平滑，开幕式将于窄。这是必要的步骤中，细胞将游离。细胞培养罩在执行消防抛光，使移液器保持无菌。 </li></ol> 2。颈上神经节的剖析： <ol><li>收集年龄P0或P1的新生幼鼠。 </li><li>迅速用70％乙醇擦拭小狗解剖，以减少污染的机会。 </li><li>快速斩首小狗一个锋利的剪刀（这一步不会显示在视频）。使用无菌钳对无菌纱布简单地漏多余的血液保持头部。 </li><li>放在头部与腹朝上，尾结束向你面向的夹层垫。割断气管，应随时可见。使用无菌注射器针头，确保头颅解剖垫以下列方式：推到垫口的屋顶虽然一针和第二针推虽然割断气管垫到。解剖显微镜下放置垫，并开始清扫。 </li><li>利用两个钳（每手之一）清除皮肤和气管周围的脂肪，小心不要去太深。这样做会暴露一双运行几乎平行于对方的气管两侧的肌肉。注：在清扫过程中，您将需要与冷无菌PBS或冷，无血清RPMI 1640培养液，用消毒，巴斯德吸管交付洗去多余的血液，以保持该地区的清洁和湿润灌溉。这可能只是一次或一次以上取决于一部作品的速度有多快。对于一个经验剥离，它需要大约只有两到三分钟，剖析了对神经节。 </li><li>使用镊子，剪切和删除一侧的肌肉。 </li><li>一旦被删除的肌肉，颈动脉会变得可见。往往是光粉红色的，含有血液。它可能会出现轻于其他血管的颜色，您可能会看到在附近，由于这个原因，颈动脉可能不太明显了。这看起来像一个“Y”形带状沿着气管和分叉动脉运行。这分岔是一个重要的里程碑，一旦它的位置，它会比较容易找到在SCG。 </li><li>在最尾端的最终割断动脉钳（另一端仍连接）稍微抬不起来。一旦你这样做，你会看到一个杏仁状，有光泽寻找组织小肿块是松散连接到动脉，只是在分叉点，THREAD像前和/或后神经节的连接词。这是颈上神经节。另一方面在使用的第二双钳轻轻分离并将其放置在一个小培养皿中包含冷，无血清RPMI 1640培养基（35毫米）。 </li><li>下一步，提取物对气管的另一端节遵循相同的协议。 </li></ol> 3。清洗神经节： <ol><li>一旦所有的神经节已被剥离出来，他们应该被释放尽可能多的多余组织。 </li><li>解剖显微镜下，您可能会看到，神经节连接件的颈内动脉，脂肪或其他组织。每手钳，工作梳理了这些不必要的材料。成无菌15毫升圆锥形，聚丙烯管清洗节，含RPMI 1640培养液。 </li><li>前和/或后神经节连接词也应削去。每个神经节神经元约10000。一旦神经元是镀金的，他们会重新长出的突起。 </li></ol> 4。建立交感神经的神经元文化： <ol><li>神经节在200xg离心2分钟，弃上清。 </li><li>重悬在0.5至1毫升0.25％胰蛋白酶溶液中的神经节。 37˚彗星水浴或培养箱中放置30分钟。这将有助于游离于神经节细胞。 </li><li> 30分钟后，添加10毫升的完全培养基，以淡化和消除胰蛋白酶和200xg离心2分钟。 </li><li>弃上清，悬浮在2毫升决赛中等。 </li><li>进一步游离于研制过程与火抛光的玻璃牧场吸管，向上和向下的神经节细胞。磨碎的神经节的20倍和管置于冰上几分钟。 </li><li>同时，火抛光的吸管更使开放窄（直径约2 / 3至1 / 2毫米）。等待几分钟，直到它冷却和磨碎的神经节的20倍以上。当你继续，将成为中期逐步多云说明这些细胞正在分离。重复此步骤，一次或两次取决于如何细胞游离。一段时间后，你会发现所有的神经节成神经细胞分离，并没有完整的神经节是在管中可见。可能有一些组织的材料，不会游离。在这一点上，让管坐了几分钟的冰，因此，任何未解离的碎片可以解决的底部。现在仔细收集从另一管的顶部和地方，几乎所有的媒体。为了确保你不收集未解离的碎片，留在试管底部约50μL。添加更多的最终介质的试管中分离的细胞：本卷取决于文化板块之一是用培养的神经元的类型。例如：如果是被聘用的24孔的菜，然后板0.5神经节（〜10,000元以及神经元），每孔0.5毫升每口井的最终中等。一个典型的大鼠垃圾由12个幼崽，这将使24神经节。这是足够的种​​子一个24孔板和终审介质的总体积为12毫升。注：神经节还包含一个人口小神经胶质细胞和其他类型的细胞，将目前的文化。在最后的媒介使用Uridine/5-FDU防止其扩散，并促进其死亡。 </li><li>饲料细胞每48小时2 / 3的介质更换新鲜的RPMI 1％马血清+ NGF和uridine/5-FDU。注意：首先，文化包含交感神经元，看起来没有突起的圆形。短突起成为解剖后可见24小时，并逐步成为密集，随着时间的推移支。 </li></ol>

<ol>
	<li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , (1998).</li><li>Biswas, S. C., Shi, Y., Sproul, A., Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-apoptotic+Bim+Induction+in+Response+to+Nerve+Growth+Factor+Deprivation+Requires+Simultaneous+Activation+of+Three+Different+Death+Signaling+Pathways.">Pro-apoptotic Bim Induction in Response to Nerve Growth Factor Deprivation Requires Simultaneous Activation of Three Different Death Signaling Pathways.</a> The Journal of Biological Chemistry. 282 (40), 29368-29374 (2007).</li><li>Bocchini, V., Angeletti, P. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nerve+Growth+Factor:+purification+as+a+30,000-molecular-weight+protein.">The Nerve Growth Factor: purification as a 30,000-molecular-weight protein.</a> Proc. Natl. Acad. Sci. USA. (64), 787-794 (1969).</li><li>Dechant, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chat+in+the+Trophic+Web:+NGF+Activates+Ret+by+Inter-RTK+Signaling.">Chat in the Trophic Web: NGF Activates Ret by Inter-RTK Signaling.</a> Neuron. 33 (2), 156-158 (2002).</li><li>Deckwerth, T., Elliott, J. L., Knudson, C. M., Johnson, E. M., Snider, W. D., Korsmeyer, 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=Bax+is+required+for+neuronal+death+after+trophic+factor+deprivation+and+during+development.">Bax is required for neuronal death after trophic factor deprivation and during development.</a> Neuron. (17), 401-411 (1996).</li><li>Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+Vitro+Studies+on+the+Nerve+Growth+Factor+(NGF)+Requirement+of+Neurons+-+I.+Sympathetic+Neurons.">Quantitative in Vitro Studies on the Nerve Growth Factor (NGF) Requirement of Neurons - I. Sympathetic Neurons.</a> Developmental Biology. (58), 96-105 (1977).</li><li>Park, D. S., Morris, E. J., Padmanabhan, J., Shelanski, M. L., Geller, H. M., Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin-dependent+kinases+participate+in+death+of+neurons+evoked+by+DNA-damaging+agents.">Cyclin-dependent kinases participate in death of neurons evoked by DNA-damaging agents.</a> The Journal of Cell Biology. 143, 457-467 (1998).</li><li>Pierchala, B. A., Ahrens, R. C., Paden, A. J., Johnson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+Growth+Factor+Promotes+the+Survival+of+Sympathetic+Neurons+through+the+Cooperative+Function+of+the+Protein+Kinase+C+and+Phosphatidylinositol+3-Kinase+Pathways.">Nerve Growth Factor Promotes the Survival of Sympathetic Neurons through the Cooperative Function of the Protein Kinase C and Phosphatidylinositol 3-Kinase Pathways.</a> The Journal of Cell Biology. 279, 27986-27993 (2004).</li></ol>

The authors have nothing to disclose.

我们用我们的文化SD大鼠。 需要时间和护理，清洗时的神经节。取出所有碎片，血管和脂肪。在确保文化，是多还是少均匀，无多余的细胞类型，这一步很重要。 uridine/5-FDU此外，将在很大程度上消除污染的文化任何剩余的非神经细胞（有丝分裂），或至少抑制其增殖。 此外，在解离步骤，耐心，花时间来单独的神经元，使他们没有发现在大的团块，一旦被接种到板块。大团块细胞不会产生良好的实?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Forceps, Stainless steel #5</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS4976</td>
<td>&nbsp;</td>
<tr>
<td>Scissors, DEAVER straight sharp-blunt- 43mm blades - 5.5</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS6760</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640 w/ L-Glutamin</td>
<td>Reagent</td>
<td>Cellgro, Mediatech, Inc.</td>
<td>10-040-CV</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>Reagent</td>
<td>SAFC Biosciences</td>
<td>12103c</td>
<td>&nbsp;</td>
<tr>
<td>Donor Horse Serum</td>
<td>Reagent</td>
<td>JRH Biosciences</td>
<td>12449</td>
<td>Heat-inactivate by incubating in 56&deg;C waterbath for 30 minutes.</td>
</tr>
<tr>
<td>Penicillin/streptomycin</td>
<td>Reagent</td>
<td>Gibco, Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin without EDTA</td>
<td>Reagent</td>
<td>Difco</td>
<td>MT 25-050-CI</td>
<td>&nbsp;</td>
<tr>
<td>Uridine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>U3003</td>
<td>&nbsp;</td>
<tr>
<td>5-Fluoro-5' deoxyuridine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>F-8791</td>
<td>&nbsp;</td>
<tr>
<td>NGF</td>
<td>Reagent</td>
<td>Harlan Bioproduct</td>
<td>BT-5025</td>
<td>&nbsp;</td>
<tr>
<td>Dissection Microscope and light source</td>
<td>Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>15 ml polypropylene tubes</td>
<td>Tool</td>
<td>BD Falcon</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>Cell culture dishes</td>
<td>Tool</td>
<td>Corning, Nunclone</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

protocol for culturing sympathetic neurons from rat superior cervical ganglia (scg)

颈上神经节（SCG）在大鼠小，有光泽，杏仁状结构，其中包含交感神经元。这些神经元提供的头部和颈部区域的交感神经支配，它们构成了一个良好的特点和人口相对同质化（4）。交感神经元是依赖于神经生长因子（NGF）的存活，分化和轴突生长和广泛的传播，神经生长因子的空房情况，有利于他们的文化和实验操作（2，3，6）。基于这些原因，培养的交感神经元，已被用于各种各样的研究，包括神经元的发育和分化，编程和病理细胞死亡的机制和信号转导（1，2，5，6）。解剖SCG从新生大鼠和培养交感神经元是不是很复杂，可以掌握相当迅速。在这篇文章中，我们将详细描述了如何剖析，从新生幼鼠在SCG并使用它们来建立培养的交感神经元。文章还将介绍预备步骤和各种试剂和实现这一目标所需要的设备。

Watch this Scientific Journal Video about Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG at JoVE.com

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

这是一个描述如何分离并培养颈上神经节（SCG）的新生幼鼠的主要交感神经元的协议。

培养大鼠颈上神经节（SCG）的交感神经元的议定书

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

protocol-for-culturing-sympathetic-neurons-from-rat-superior-cervical

Research

JoVE Journal

Biology

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

22.6K 次观看.  Columbia University. 这是一个描述如何分离并培养颈上神经节（SCG）的新生幼鼠的主要交感神经元的协议。

视频: Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

准备在微流体装置E18条块皮质大鼠神经元

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

科研

生物学

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

嗜中性粒细胞的分离协议

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

隔间文化背根神经节神经元的制备及维修

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

蠕虫病毒实验室介绍：从培养蠕虫突变

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

一个简单的协议提取血细胞从野生毛毛虫

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

原代培养大鼠内髓集合管细胞

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

从大鼠的侧尾静脉血液取样

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

CD146的分离<sup&gt; +</sup&gt;从大鼠肺组织驻地肺间质基质细胞

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

胰腺胰岛与小鼠分离的简单高效协议

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergstr&#246;m technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

This technical report describes a variation of the modified Bergstr&#246;m technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

从人类肌肉提比利斯前部高级隔间收集骨骼肌肉活检，用于机械评估

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...