这项工作得到了NIH的OD R24拨款OD031956和NICHD R01拨款HD073104的支持。我们感谢达西·凯利（Darcy Kelly）对该协议的有益讨论和初步意见。我们还要感谢Samantha Jalbert，Jill Ralston和Wil Ratzan的帮助和支持，以及我们的三位匿名同行评审员的反馈。

所有实验均按照哈佛医学院IACUC（机构动物护理和使用委员会）（IS 00001365_3）的规则和规定进行。
注意：尽管美国兽医协会12认为所描述的主要安乐死方法是一种可接受的安乐死技术，但尚未发现它会导致心跳停止13。即使是常用的二次双皮管方法也不能阻止这种情况，也不能从动物身上取出心脏。麻醉动物的放血被认为是成功安乐死的人道和有效的方法12.由于通过安乐死维持新鲜组织是该协议的目标，因此心脏继续通过MS-222的初级安乐死跳动是有益的，并且灌注本身是通过放血的继发性安乐死方法。
1. 准备
<ol><li>确保研究机构已批准本协议中描述的安乐死和灌注技术。</li>
	<li>制备5 g / L MS-222（甲磺酸三卡因）和5 g / L碳酸氢钠的溶液。体积应大于完全覆盖被安乐死动物所需的体积。检查pH值以确保其为≥7。</li>
	<li>每乳杆菌制备 500 μL 180 U/mL 肝素的磷酸盐缓冲盐水 （PBS），或每热带乳杆菌制备 200 μL。</li>
	<li>通过将 非洲爪蟾 放入该溶液中进行初级安乐死（从步骤1.2开始）;动物将保持浸没状态总共1小时。</li>
	<li>确认 非洲爪蟾 在安乐死后 15 分钟捏住脚失去了疼痛反应。如果动物有反应，请将其返回到安乐死溶液中，直到失去这种反应。</li>
	<li>称量非洲爪蟾并在取样前进行所需的任何其他测量。</li>
	<li>使用 31 G 针头，将 250 μL 的 X. laevis 和 100 μL 180 U/mL 肝素的 PBS（来自步骤 1.3）注射到每个前肢的肌肉组织中。</li>
	<li>在PBS灌注液中制备1mL / g动物重量的54U / mL肝素溶液。对该方案更有经验的人可能会发现完成灌注所需的培养基更少。</li>
	<li>使用 22 G 皮下注射针头灌注 X. laevis，使用 25 G 皮下注射针头灌注 热带弧菌。用线切割器修剪尖端，使灌注针变钝（图1）14。 注意：如果移动，这降低了针头穿过心室的可能性。除了钝化外，针头可能会用磨刀石或锉刀稍微磨碎，但仍保持足够锋利以刺穿心室。</li>
	<li>通过连接修剪好的针头并循环 54 U/mL 肝素化 PBS 灌注物（从步骤 1.8 开始）来准备泵。确保清除管道中的所有气泡，以消除空气栓塞的可能性，这会导致灌注效率降低或失败（见 表1）。在手术过程中将灌注介质保持在冰上。</li>
	<li>如果泵不可编程，请在针头就位的情况下，测量不同设置下的泵送介质体积，以确定哪些设置最接近 5 mL/min 和 10 mL/min。无论物种如何，都将使用这些流速。如果灌注泵是可编程的，请按照制造商的说明使用针头进行校准。</li>
	<li>将解剖表面（托盘或泡沫板）放在辅助容器内的斜坡处，或将其布置以方便血液引流。</li>
	<li>一旦青蛙在溶液中放置1小时，初级安乐死就完成了。取出青蛙，并通过捏脚重新检查疼痛反应的丧失。</li>
	<li>将青蛙放在背部并固定每个肢体（图2）。如果需要保留四肢组织，可以通过四肢周围的手指或 U 形订书钉放置细针。</li>
	<li>用解剖剪刀，切开皮肤，沿着中线向上，然后横向，做两个皮瓣。（图2）</li>
	<li>使用镊子抓住白线并将其从腔腔中拉开（图3）。小心地用剪刀剪开肌肉组织。从腔壁上制作两个襟翼，并将所有襟翼切开或固定。</li>
	<li>使用解剖剪刀切开喙骨并切掉多余的组织，以更好地进入心脏（图3）。</li>
	<li>心脏应该还在跳动。如果心脏在灌注前停止跳动，请注意样品新鲜度已受到影响。</li>
</ol>2. 灌注
<ol><li>识别胃并轻轻移动它，使其位于肝脏左叶的顶部（在观察者的右侧），其脉管系统在手术过程中可见。识别肺并使用组织钳抓住它的尖端。将肺拉出腔腔外并将其固定在尖端（图4）。轻轻地这样做，因为破裂的血管不能很好地灌注。注意叶内是否可见血液，因为这会影响确定手术完成的能力。</li>
	<li>拍摄腔腔的图像，以更好地评估灌注效率，并可能在以后识别异常组织。</li>
	<li>识别薄心包并用组织钳将其拉动（图5）。使用虹膜切除剪刀的尖端轻轻穿孔心包，注意不要切割下面的组织。将心包从心脏的三个腔室剥离。</li>
	<li>用镊子轻轻抓住心室的顶点。施加有限的压力，以便在镊子的牵引表面之间有足够的空间供灌注针通过（图6）。</li>
	<li>通过镊子的闭合将针头插入心室腔室，注意不要穿过心室（图7）。使用止血器使用持针器将组织钳夹到位。 注意：此技术可稳定针的位置，该位置仍然锋利。将针头直接夹在心室上也会造成不必要的损伤，如果需要重新夹紧，则使重新夹紧更加困难（见 表1）。</li>
	<li>以大约 5 mL/min 的速度启动泵的流量。心脏和动脉干的三个腔室会充血（图8;见 表1）。</li>
	<li>用剪刀小心地用右耳廓（在观看者的左侧）;鲜血会涌出。将流速设置为 5 mL/min 或增加到 10 mL/min。</li>
	<li>继续直到胃的脉管系统变白（见 表1），然后刮起心脏的左耳廓（在观察者的右侧）。如果流速仍为 5 mL/min，请将其增加到大约 10 mL/min。</li>
	<li>使用移液管冲洗灌注介质中的腔腔，以帮助保持可见性并更好地评估从耳廓流出的灌注液的颜色。</li>
	<li>将针保持在原位，直到从耳廓流出的灌注液清除（见 表1）并且肺失去红色（见 表1; 图9）。</li>
</ol>

用于转录组学和蛋白质组学的非洲爪蛙血液灌注

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(124), e55740 (2017).</li><li>Lametschwandtner, A., Minnich, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+anatomy+of+the+brain+of+the+adult+pipid+frog,+Xenopus+laevis+(Daudin):+A+scanning+electron+microscopic+study+of+vascular+corrosion+casts.">Microvascular anatomy of the brain of the adult pipid frog, Xenopus laevis (Daudin): A scanning electron microscopic study of vascular corrosion casts.</a> Journal of Morphology. 279 (7), 950-969 (2018).</li><li>Lametschwandtner, A., Minnich, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+anatomy+of+the+urinary+bladder+in+the+adult+African+clawed+toad,+Xenopus+laevis:+A+scanning+electron+microscope+study+of+vascular+casts.">Microvascular anatomy of the urinary bladder in the adult African clawed toad, Xenopus laevis: A scanning electron microscope study of vascular casts.</a> Journal of Morphology. 282 (3), 368-377 (2021).</li><li>Lametschwandtner, A., 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=Microvascular+anatomy+of+the+gallbladder+of+the+adult+South+African+clawed+toad,+Xenopus+laevis+Daudin:+A+scanning+electron+microscope+study+of+vascular+corrosion+casts.">Microvascular anatomy of the gallbladder of the adult South African clawed toad, Xenopus laevis Daudin: A scanning electron microscope study of vascular corrosion casts.</a> Microscopy and Microanalysis. 13, 492-493 (2007).</li><li>Lametschwandtner, A., Spornitz, U., Minnich, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+anatomy+of+the+non-lobulated+liver+of+adult+Xenopus+laevis:+A+scanning+electron+microscopic+study+of+vascular+casts.">Microvascular anatomy of the non-lobulated liver of adult Xenopus laevis: A scanning electron microscopic study of vascular casts.</a> Anatomical Record. 305 (2), 243-253 (2022).</li><li>Miodo&#324;ski, A. J., B&#228;r, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+supply+of+the+choriocapillaris+of+anuran+amphibians+(Rana+temporaria,+Rana+esculenta).+Scanning+electron-microscopic+(SEM)+study+of+microcorrosion+casts.">Arterial supply of the choriocapillaris of anuran amphibians (Rana temporaria, Rana esculenta). Scanning electron-microscopic (SEM) study of microcorrosion casts.</a> Cell and Tissue Research. 249 (1), 101-109 (1987).</li><li>Nenni, M. J., 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=Xenbase:+Facilitating+the+use+of+Xenopus+to+model+human+disease.">Xenbase: Facilitating the use of Xenopus to model human disease.</a> Frontiers in Physiology. 10, 154 (2019).</li><li>Tandon, P., Conlon, F., Furlow, J. D., Horb, M. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+physiology+and+diseases+of+amphibians.+Veterinary+clinics+of+North+America.">Cardiovascular physiology and diseases of amphibians. Veterinary clinics of North America.</a> The Veterinary Clinics of North America. Exotic Animal Practice. 12 (1), 39-50 (2009).</li><li>Stephenson, A., Adams, J. 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作者声明没有竞争利益。

该协议描述了用于进入腔腔的传统解剖技术。其他技术也是可以接受的，只要它们对组织造成的损害最小，心脏是可接近的，肺和胃是可见的。同样，列出的大多数解剖工具都可以很容易地用类似的项目代替。
虽然已经尝试优化该程序的功效，但结果可能会因一个人的经验和个体青蛙之间的变异性而异。血液灌注的一个有趣的方面仍然不在本文的范围之内，即该程序与接受?...

为了保存和固定的目的，常规完成两栖动物的全身灌注1，2，3，4，5，6。然而，这些程序的发生速度限制了每只动物可以采集的新鲜样本数量。这项工作的目标是在非洲爪蟾中开发一种有效的血液灌注方案，优先考虑该技术的速度。对于热带X. laevis，每只动物需要不到10分钟，每只X. laevis动物需要不到15分钟。次要优先事项是易于复制和使用易于获取的设备，以便可以在非洲爪蟾实验室之间广泛共享高质量的样品。
非洲爪 蟾广泛用于生物医学研究，以研究跨物种保存的基本生物学和病理过程。这种四足动物与其他水生模型相比，与哺乳动物有更密切的进化关系，有肺、三腔心脏和带手指的四肢。国际社会有效地利用 非洲爪蟾 ，通过对疾病相关基因功能的深入疾病建模和分子分析，对人类疾病有了更深入的了解。 非洲爪蟾 作为动物模型的众多优点使其成为研究人类发育和疾病的分子基础的宝贵工具;这些优点包括：卵母细胞和胚胎大小大，繁殖力高，易于容纳，快速的外部发育以及易于基因组操作。据估计，非洲 爪蟾 共享~80%的已确定的人类疾病基因7。
与流行的哺乳动物模型相比，非洲爪蟾是一种快速、经济高效的模型，具有吗啉蛋白敲低的易用性，并且可以使用CRISPR8进行高效的转基因和靶向基因突变。定量质谱和单细胞转录组学已成功应用于非洲爪蟾胚胎9，10，但最近来自非洲爪蟾的细胞图谱显示，大多数组织的组成以血细胞11型为主。通过开发一种快速放血组织并使用冷却培养基的技术，样品新鲜度受灌注的影响最小。这对于以分析生理上不受干扰的mRNA或蛋白质表达为目标的应用尤其重要。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5x Magnifying glass with LED light and stand</td>
			<td>amazon.com</td>
			<td>B08QJ6J8P1</td>
			<td>light must not produce heat</td>
		</tr>
		<tr>
			<td>Disposable transfer pipets</td>
			<td>VWR</td>
			<td>414004-036</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting fine-pointed forceps</td>
			<td>Fisher Scinetific</td>
			<td>08-875</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors sharp piont, straight 6.5&#34;</td>
			<td>VWR</td>
			<td>76457-374</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection tray</td>
			<td>Fisher Scinetific</td>
			<td>14-370-284</td>
			<td>styrofoam sheets are an acceptable alternative</td>
		</tr>
		<tr>
			<td>Euthanasia container</td>
			<td>US Plastic</td>
			<td>Item&#160;2860</td>
			<td>alternative opaque containers acceptable</td>
		</tr>
		<tr>
			<td>Euthanasia container lid</td>
			<td>US Plastic</td>
			<td>Item&#160;3047</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine dissection pins</td>
			<td>Living Systems Instrumentation</td>
			<td>PIN-#3</td>
			<td></td>
		</tr>
		<tr>
			<td>General use hypodermic needles, 22 G</td>
			<td>Fisher Scientific</td>
			<td>14-826-5A</td>
			<td>for X. laevis</td>
		</tr>
		<tr>
			<td>General use hypodermic needles, 25 G</td>
			<td>Fisher Scientific</td>
			<td>14-826AA</td>
			<td>for X. tropicalis</td>
		</tr>
		<tr>
			<td>Heparin, porcine intestinal mucosa</td>
			<td>MilliporeSigma</td>
			<td>37-505-410MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Iridectomy scissors 6&#34;</td>
			<td>vwr</td>
			<td>470018-938</td>
			<td>iris scissors are an acceptable alternative</td>
		</tr>
		<tr>
			<td>Luer-to-barb adapter male Luer with lock ring</td>
			<td>amazon.com</td>
			<td>B09PTX6M2Z</td>
			<td>size will be dependant on the hosing of the pump used</td>
		</tr>
		<tr>
			<td>Mayo-Hegar needle holder</td>
			<td>Fisher Scinetific</td>
			<td>08-966</td>
			<td>mosquito forceps are an acceptable alternative</td>
		</tr>
		<tr>
			<td>MS-222: Syncaine (formerly tricaine)</td>
			<td>Pentair AES</td>
			<td>TRS1</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic liquid pump dosing pump 5&#8211;100 mL/min</td>
			<td>amazon.com</td>
			<td>B07PWY4SM6</td>
			<td>any peristaltic pump capable of pumping 5-10mL/min is acceptable</td>
		</tr>
		<tr>
			<td>Sharpening stone</td>
			<td>VWR</td>
			<td>470150-112</td>
			<td>optional; for dulling needles</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate, powder, USP</td>
			<td>Fisher Scientific</td>
			<td>18-606-333</td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen forceps, serrated</td>
			<td>VWR</td>
			<td>82027-442</td>
			<td></td>
		</tr>
		<tr>
			<td>T-Pins for dissecting</td>
			<td>Fisher Scinetific</td>
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effective rapid blood perfusion in xenopus

100多年来，非洲爪蟾一直是了解脊椎动物发育和疾病的强大模式生物。在这里，定义了非洲爪蟾的快速血液灌注方案，旨在一致和急剧地减少所有组织内的血液。通过将针头直接插入心脏心室并通过血管系统泵送肝素化磷酸盐缓冲盐水（PBS）来进行灌注。每只动物可以在大约 10 分钟内完成该过程。血液由一些高度丰富的蛋白质和细胞类型主导，由于这些蛋白质掩盖了大多数其他感兴趣的分子和细胞类型，因此会产生许多问题。使用定量蛋白质组学和单细胞转录组学对成人非洲爪蟾组织的可重复表征将受益于在器官取样之前应用该协议。组织取样的方案在配套论文中定义。这些程序旨在使不同性别、年龄和健康状况的非洲爪蟾的做法标准化，特别是非洲爪蟾和热带爪蟾。

成功灌注后，所有组织（色素沉着爪蟾中的肝脏除外）将明显变浅，血液饱和度较低。主要血管将变得不那么明显（图10），取样后组织（不包括肝脏）将在缓冲液中干净冲洗。虽然该方案的成功执行最终只能通过出血组织样本的数据质量来确认，但故障排除表中提供了几个典型问题、可能的原因和建议的补救措施（表1和图11<...

Watch this Scientific Journal Video about Effective Rapid Blood Perfusion in Xenopus at JoVE.com

作者聚焦：非洲爪蟾中的有效快速血液灌注  

这里介绍的是一种有效的快速血液灌注方案，用于制备非洲爪蛙的组织样本，用于转录组学和蛋白质组学研究。

非洲爪蟾的有效快速血液灌注

Xenopus have been powerful model organisms for understanding vertebrate development and disease for over 100 years. Here, a rapid blood perfusion protocol ...

该程序以以最大化效率的方式人道地利用两栖动物模型为中心。标准化实践可以应用于在原产地采集和保存的样本，从而减轻生物安全风险并消除动物运输压力。浓缩可以快速完成，最大限度地延长了在组织新鲜时取样的时间。这种新鲜度对于蛋白质组学和转录组学分析尤为重要。该协议详细而明确，使其可用于具有各种背景的个人。这有助于非洲爪蟾实验室之间共享高质量样品。除了这种丰富的技术外，正在制定一项类似的综合方案，用于所有主要器官的标准化取样。

effective-rapid-blood-perfusion-in-xenopus

Research

JoVE Journal

Biology

Effective Rapid Blood Perfusion in Xenopus

4.3K 次观看.  Harvard Medical School. 该程序以以最大化效率的方式人道地利用两栖动物模型为中心。标准化实践可以应用于在原产地采集和保存的样本，从而减轻生物安全风险并消除动物运输压力。浓缩可以快速完成，最大限度地延长了在组织新鲜时取样的时间。这种新鲜度对于蛋白质组学和转录组学分析尤为重要。该协议详细而明确，使其可用于具有各种背景的个人。这有助于非洲爪蟾实验室之间共享?...

视频: 作者聚焦：非洲爪蟾中的有效快速血液灌注  

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

从非洲爪蟾女性获得卵子

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

科研

生物学

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

在使用分馏爪蟾卵提取物的体外核大会

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

非洲爪蟾卵母细胞的微量注射

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

可视化的RNA中的本地化爪蟾卵母细胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

利用磁调制生物传感系统的生物检测的快速均质检测

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

在面部移植<em&gt;非洲爪蟾</em&gt;胚胎

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

该<em&gt;爪蟾</em&gt;卵母细胞切割开凡士林隙电压钳技术与荧光

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...