我们感谢弗吉尼亚Doherty和俊美姚明的技术援助。该研究是由镍氢电池（R01 MH084966），和美国陆军研究办公室和国防高级研究计划局（授予W911NF-10-1-0059）到KAG。

是用带成年雄性Long-Evans大鼠全部实验。所有的程序都按照健康（NIH）指南实验动物的护理和使用美国国家研究院​​和批准了麻省理工学院的机构动物护理和使用委员会及动物护理和使用审查办公室该USAMRMC。 1.准备<ol><li>通过在针放置屏蔽针在含肝素500微升管（1000 USP单位/ ml），然后抽吸和排出肝素溶液Heparinise导管和注射器。 <ol><li>附加蝴蝶导管注射器。保持屏蔽过导管，以保护该尖端从损坏的针。 </li><li>撤回肝素的体积比的血液将被收集的体积稍大。取下注射器，并用空气填充它。 </li><li>重新连接的注射器的导管，并使用空气排出体内多余的肝素液;确保只有微量残留在管，针头，和注射器。 </li><li>将无菌导尿管，用还连着注射器，在无菌的表面。 </li></ol></li><li>快速安全的老鼠在一个干净的布，确保fore-和后爪有一个舒服的姿势和呼吸不受限制。 <ol><li>与固定钩环带的包;确保外生殖器不收缩。 </li><li>有一个助手轻轻地，坚决抑制大鼠（腹部和尾部的基地）上扎实的工作表面用尾巴挂在柜台边。 </li></ol></li></ol> 2.采血<ol><li>浸泡在42℃的水中尾巴40-50秒，以扩张血管，并用干纸巾的尾巴。找到尾静脉放血（同尾转动整个身体，以防止扭曲的尾巴）。 注:尾巴的充分变暖的快速收藏品关键为n的血液试样。如果脉管收缩，导管的正确放置是困难的，并且血流量大大减少。加热垫可被用作替代水浸泡。 </li><li>确定的采样点。 注意:动脉位于沿尾部的中间背侧;不要使用它进行抽样。 <ol><li>目标要么位于横向于动脉的左，右尾静脉。尾部，这由各个应变和随着年龄的增加色素沉着，可能会掩盖一些血管。在尾部的下部定位的静脉的一部分。 </li><li>擦拭用2％氯己定的防腐溶液中的目标区域。 </li></ol></li><li>通过抽出从零柱塞到约50微升创建在注射器和导管的负压。 </li><li>轻轻地紧紧握住尾巴尖端附近，以保持尾部直线整个样本采集。确保血液流动不被过度得紧闭塞。</li><li>慢慢将导管插入静脉以浅角度从尾尖约5厘米。当静脉被穿透，血液将流入导管。慢慢撤回注射器的柱塞以收集在一个稳定的速率所需要的体积（〜每秒20微升）。 <ol><li>请教兽医人员约可收集的最大血量的信息。血应当收集的最大量取决于大鼠的体重和健康状况。别在14天时间段撤回总血量的15％以上。 注:血液是更加困难的动物都敏锐地强调抽样集合，因为应激激素收缩血管的前几分钟，收集。例如，移动老鼠的家笼，以一种新颖的房间，需要几分钟来包装动物，或在导管的重复插入到静脉都可能引发急性应激反应。 </li><li>促进布卢通过"挤奶"静脉d流。运行一个手指沿静脉的长度，从朝向尾部的末端的基础上，但仍超过2厘米从插入针或导管的尖端可以成为从静脉移位。 </li><li>如果血液不能成功地从导管渗透的初始部位采集，还重新插入针上的静脉。如果收集血液在最初部位，通过断开并重新连接导管和注射器重新插入静脉前重新加压针头。在一般情况下，避免额外的穿透。 </li><li>作为多个穿透可导致尾静脉塌陷，其中的血液供应的尾部被切断和软尾组织变得坏死，安乐死大鼠是否有尾静脉塌陷。 </li></ol></li><li>当足够的样品体积被收集，通过断开和重新连接该导管释放压力的注射器。稍微吸用注射器活塞（约50＆＃181;升），并撤回从静脉针头。 注意:如果针不首先释放压力的注射器抽出，血液滴落从针。 </li><li>简要施加压力插入部位止血，并用擦拭消毒液的区域。返回老鼠的笼子。 </li></ol> 3.处理血样<ol><li>吸空气，以确保没有血液保持导管针内，并用剪刀剪开的导管就在上面的针。排出血液进入无菌1.5 ml离心管。 注意:如果血液通过针推动时，所述剪切力可能导致红血细胞破裂可以与许多下游测定干扰。卸下针以避免溶血。 <ol><li>收集血浆，含有EDTA作为抗凝剂（这里使用管，用10微升的0.1M EDTA为200-400微升血液;确保EDTA的使用不干扰瓦特的浓度第i个下游测定）和在冰上的地方。 <ol><li>在冷冻离心机（4℃）内收集的10分钟10分钟旋转的全血样品在2100×g离心。洗脱的血浆，避免干扰的红，白血细胞层。 </li></ol></li><li>收集血清，代替样品（无抗凝血剂）在室温下长达30分钟，以使凝块。在2000×g下旋转该收集管在冷冻离心机（4℃）。血清然后可以洗脱。 </li></ol></li><li>使用立即样品或储存在-80℃下长达一年。 </li></ol>

<ol>
	<li>Vausort, M., Wagner, D. R., Devaux, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+Noncoding+RNAs+in+Patients+with+Acute+Myocardial+Infarction.">Long Noncoding RNAs in Patients with Acute Myocardial Infarction.</a> Circ Res. 115 (7), 668-677 (2014).</li><li>Shah, R., 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=Biomarkers+for+Early+Detection+of+Colorectal+Cancer+and+Polyps:+Systematic+Review.">Biomarkers for Early Detection of Colorectal Cancer and Polyps: Systematic Review.</a> Cancer Epidemiol Biomarkers Prev. 23 (9), 1712-1728 (2014).</li><li>Chan, M. K., 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=Applications+of+blood-based+protein+biomarker+strategies+in+the+study+of+psychiatric+disorders.">Applications of blood-based protein biomarker strategies in the study of psychiatric disorders.</a> Prog Neurobiol. , (2014).</li><li>Cao, L., Li, L., Lin, D., Zuo, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+induces+learning+impairment+that+is+mediated+by+interleukin+1beta+in+rodents.">Isoflurane induces learning impairment that is mediated by interleukin 1beta in rodents.</a> PLoS One. 7 (12), e51431 (2012).</li><li>Culley, D. J., Baxter, M. G., Yukhananov, R., Crosby, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+impairment+of+acquisition+of+a+spatial+memory+task+following+isoflurane-nitrous+oxide+anesthesia+in+rats.">Long-term impairment of acquisition of a spatial memory task following isoflurane-nitrous oxide anesthesia in rats.</a> Anesthesiology. 100 (2), 309-314 (2004).</li><li>Vahl, T. P., 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=Comparative+analysis+of+ACTH+and+corticosterone+sampling+methods+in+rats.">Comparative analysis of ACTH and corticosterone sampling methods in rats.</a> Am J Physiol Endocrinol Metab. 289 (5), E823-E828 (2005).</li><li>Kalinichenko, L. S., Koplik, E. V., Pertsov, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+profile+of+peripheral+blood+in+rats+with+various+behavioral+characteristics+during+acute+emotional+stress.">Cytokine profile of peripheral blood in rats with various behavioral characteristics during acute emotional stress.</a> Bull Exp Biol Med. 156 (4), 441-444 (2014).</li><li>McEwen, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+effects+of+stress+hormones+in+health+and+disease:+Understanding+the+protective+and+damaging+effects+of+stress+and+stress+mediators.">Central effects of stress hormones in health and disease: Understanding the protective and damaging effects of stress and stress mediators.</a> Eur J Pharmacol. 583 (2-3), 174-185 (2008).</li><li>Sanchez, A., Toledo-Pinto, E. A., Menezes, M. L., Pereira, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+norepinephrine+and+epinephrine+concentrations+in+adrenal+gland+of+the+rats+submitted+to+acute+immobilization+stress.">Changes in norepinephrine and epinephrine concentrations in adrenal gland of the rats submitted to acute immobilization stress.</a> Pharmacol Res. 48 (6), 607-613 (2003).</li><li>Meyer, R. M., Burgos-Robles, A., Liu, E., Correia, S. S., Goosens, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ghrelin-growth+hormone+axis+drives+stress-induced+vulnerability+to+enhanced+fear.">A ghrelin-growth hormone axis drives stress-induced vulnerability to enhanced fear.</a> Mol Psychiatry. , (2013).</li><li>Knoll, A. T., Carlezon, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynorphin,+stress,+and+depression.">Dynorphin, stress, and depression.</a> Brain Res. 1314 (56-73), (2010).</li><li>Harvey, B. H., Brand, L., Jeeva, Z., Stein, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical/hippocampal+monoamines,+HPA-axis+changes+and+aversive+behavior+following+stress+and+restress+in+an+animal+model+of+post-traumatic+stress+disorder.">Cortical/hippocampal monoamines, HPA-axis changes and aversive behavior following stress and restress in an animal model of post-traumatic stress disorder.</a> Physiol Behav. 87 (5), 881-890 (2006).</li></ol>

作者什么都没有透露。

在这里，我们描述了用于获得从大鼠其中比其它常用的技术提供显著优点的血液样品的快速和简单的程序。第一，它不需要麻醉，在与从颈静脉或眶后窦抽样对比。当被收集的血液样本周边的行为过程，麻醉剂的给药是不利的，因为它可以与学习和记忆4,5干扰。第二，它提供了收集较大血液体积比其他静脉穿刺技术，例如收集从隐或足背静脉的能力。使用此处所描述的，技术可达1.5毫升的血可从大鼠收集在一个单一的时间点，一个容积容易允许多个测定以并行运行。最后，此过程最大限度地减少组织损伤相比，尾尖截肢或眶后出血的可能性。使用这种方法的促进符合动物W¯¯elfare法案和指南的护理和使用实验动物 ，需要尽量减少所造成的对动物进行实验过程的痛苦和困扰。 建议在调查新的这个方法练习约束和尾部出血技术，以最小化该实验动物被约束的时间。血液从约束的开始收集在不到3分钟提供最佳结果。 此处所描述的协议可被用于采样每周1至4次，但不超过每天两次以上。而反复血液采集，可以执行不同的采样点从尾的基部向上移动应用，和左，右尾静脉应交替作为采样点。啮齿动物的血液总体积是其体重的6-7％，而总血量的不超过15％应为2周的时间内进行收集。血清或等离子体包括所收集的样品体积的约40-60％。 经由侧尾静脉血液取样，也可以在小鼠所描述这里有一些小的修改进行的。首先，可以仅使用小计（27 G）的导管。其次，它是推荐使用的管限制器，而不是一个包装，以固定的小鼠。血液可从使用颌下维管束（200-500微升）的静脉穿刺的小鼠中得到的体积大于可以从尾静脉（200微升最大）被安全地收集。因为从颌下维管束采血需要最少的约束，并且可以得到更多的血液，这是对于采样在小鼠的优选途径。 与该程序可以被执行，以及它的微创性质的快速性，也由急性应激反应6最小化的基于血液的措施的潜在扰动。该急性应激反应可以改变许多分子的循环水平，包括白细胞介素和其它免疫活性因子7，下丘脑-垂体-肾上腺轴8的激素，在交感神经系统9激素，生长激素释放肽10，内源性阿片11，多巴胺和血清素12。如果这些分子或其他由这些分子调节的静止循环措施都需要，以减少应激反应，其内为小触发作为应激曝光开始的一分钟内是很重要的。 应激反应不仅改变血液的组合物，也代表对血液取样，因为脉管的狭窄的一个技术障碍经由从交感神经系统增加驱动器。它变得越来越难以得到从正在安装一种急性应激反应的老鼠稳定的血流量。因此，动物的痛苦必须是微量源化为了迅速得到反映感兴趣的生理状态的样品。

从血液获得的生物标志物提供有用的诊断，预测，并分层在许多临床环境的措施，包括心血管疾病1，癌症科学2，和精神疾病3。它们也可用于在基础科学评估生物的"状态"，包括饥饿，炎症，或应力存在的程度。这种措施可以通过变量，可能会或可能不会与感兴趣的问题，包括一天中所获得的样品的时间和受试者的性别临界的影响。它也可以通过在血液取样程序本身产生的应力的影响。应激激素和疼痛的感觉可迅速改变血液的组合物。 啮齿动物是最常用的实验室动物，和多种方法已经被开发用于采血。血液取样的理想方法应当具有最小的生理学对动物的影响升，无需麻醉，允许快速和反复取样，并为众多的下游应用提供足够的血量。流行的技术，用于收集血液，如颈静脉或尾尖截肢不符合这些标准的导尿。 此协议的目的是描述在大鼠中是最低限度的压力为使用一个血液采样技术，不需要麻醉，允许在单个受试者的多个血液采集，并提供了这样的多个测定可进行相对大的样本体积在单个样本。该方法的目标是获得了由急性应激反应最小的影响的血液样本。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium heparin (1000 USP units/ml)</td>
			<td>Patternson Veterinary Supply</td>
			<td>25021040010</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>JT Taylor</td>
			<td>JT2020-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dermachlor Rinse-Chlorhexadine</td>
			<td>Butler Schein</td>
			<td>6356</td>
			<td>Topical antiseptic solution, 2% chlorhexidine gluconate</td>
		</tr>
		<tr>
			<td>SURFLO Winged Infusion Sets, Terumo, butterfly catheters</td>
			<td>VWR Scientific</td>
			<td>TESV25BLK</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Tuberculin 1cc syringes</td>
			<td>VWR Scientific</td>
			<td>BD309659</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge tubes</td>
			<td>VWR Scientific</td>
			<td>89202-682</td>
			<td></td>
		</tr>
		<tr>
			<td>500 &#956;l microcentrifuge tubes</td>
			<td>VWR Scientific</td>
			<td>21150-330</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors, stainless steel, 5&#34;</td>
			<td>VWR Scientific</td>
			<td>82027-586</td>
			<td></td>
		</tr>
		<tr>
			<td>500ml plastic beaker</td>
			<td>VWR Scientific</td>
			<td>414004-149</td>
			<td></td>
		</tr>
		<tr>
			<td>Clean cloth wrap</td>
			<td>Butler Schein</td>
			<td>2993</td>
			<td></td>
		</tr>
		<tr>
			<td>Velcro tape, .75&#34; width</td>
			<td>Monoprice</td>
			<td>B004AF9II6</td>
			<td>Hook and loop tape</td>
		</tr>
		<tr>
			<td>Timer</td>
			<td>VWR Scientific</td>
			<td>62344-641</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

从作为在协议中所述的横向尾静脉采集血液的血浆给出的血浆样品，这是半透明的，并在外观淡黄色。 如图1，溶血的样品中赋予红色色彩到等离子体。急性应激反应能迅速地改变血液的组合物。例如，循环皮质酮浓度可以在应激曝光10分钟显着增加，如图2中所示。皮质酮与对应激曝光之前该方法得到的低基础水平表明，采样过程本身不是压力的显著源。 <img alt="图1" src="/files/ftp_upload/52766/52766fig1.jpg" /> 图1:样品的外观（A）溶血样品显示。离心后，将血浆或血清层（表面表示以黑色芳行）似乎略带粉红色或红色。暗色调表明溶血更高水平。 （B）的离心分离后，一个适当的收集的样品将有一个明确的，淡黄色的外观上频带（表面由黑色箭头表示），其对应于非溶血的血浆或血清。除去该层时，它不干扰底层的全血，或者通过推压枪头进入全血层或通过吸一些全血进入前端是重要的。沾染全血任何血浆或血清应该被丢弃。 <img alt="图2" src="/files/ftp_upload/52766/52766fig2.jpg" /> 图2:血浆皮质酮迅速升高承接压力的经验血从侧尾静脉成年雌性Long-Evans大鼠的所得前和10分钟，在暴露于4种色调（10秒，2千赫，85 dB为单位）共同终止。以footshocks（1秒，350微安）。血浆皮质酮基线（290.4±138.8微克/毫升）显著小于水平观察到10分钟以下介绍的电击应力的（2204.8±454.5微克/毫升，p值= 0.02中，n = 4），如通过配对t确定 - 试验。 *，P &lt;0.05

Watch this Scientific Journal Video about Sampling Blood from the Lateral Tail Vein of the Rat at JoVE.com

Sampling Blood from the Lateral Tail Vein of the Rat

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, ...

sampling-blood-from-the-lateral-tail-vein-of-the-rat

Research

JoVE Journal

Biology

102.3K 次观看.  Massachusetts Institute of Technology. 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.

视频: Sampling Blood from the Lateral Tail Vein of the Rat

Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: retro-orbital sinus, jugular vein, maxillary vein, saphenous vein, heart. Each technique has its advantages and disadvantages, and some are not approved any more in some countries (e.g., retro-orbital draws in Holland). A discussion of different techniques for drawing blood are available 1-3.
Here, we present two techniques for drawing blood from rats, each with its specific applications.
Blood draw from the saphenous vein, provided it is done properly, induces minimal distress in animals and does not require anesthesia. This technique allows repeated draws of small amounts of blood, such as needed for pharmacokinetic studies 4,5, determining plasma chemistry, or blood counts 6.
Cardiac puncture allows the collection of large amounts of blood from a single animal (up to 10 ml of blood can be drawn from a 150 g rat). This technique is therefore very useful as a terminal procedure when drawing blood from the saphenous would not provide a large enough sample. We use cardiac puncture when we need sufficient amounts of serum from a specific strain of rats to grow T lymphocyte lines in vitro 4-9.

Blood draws are necessary in a large number of studies, for example to study the pharmacokinetics profile of a compound. Here, we demonstrate how to draw blood from rats using two techniques: blood draw from the saphenous vein or by cardiac puncture.

从老鼠血液通过大隐静脉和心脏穿刺“

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: ...

科研

生物学

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

从特定的大脑区域的RNA或蛋白质提取大鼠脑微解剖

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

从美国的马蹄蟹，鲎波吕斐摩斯采血车

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

制备鼠尾肌腱的生物力学和Mechanobiological研究

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...

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 ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

在离体血液灌注大鼠肺备量化单微血管通透性

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Non-Terminal Blood Sampling Techniques in Guinea Pigs

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently represents a relatively narrow area of research and descriptive data on specific methodology is correspondingly scarce. The anatomical features of guinea pigs are slightly different from other rodent models, hence modulation of sampling techniques to accommodate for species-specific differences, e.g., compared to mice and rats, are necessary to obtain sufficient and high quality samples. As both long and short term in vivo studies often require repeated blood sampling the choice of technique should be well considered in order to reduce stress and discomfort in the animals but also to ensure survival as well as compliance with requirements of sample size and accessibility. Venous blood samples can be obtained at a number of sites in guinea pigs e.g., the saphenous and jugular veins, each technique containing both advantages and disadvantages4,5. Here, we present four different blood sampling techniques for either conscious or anaesthetized guinea pigs. The procedures are all non-terminal procedures provided that sample volumes and number of samples do not exceed guidelines for blood collection in laboratory animals6. All the described methods have been thoroughly tested and applied for repeated in vivo blood sampling in studies within our research facility.

Though a known model, the guinea pig currently represents a niche in experimental animal sciences and limited data is available on the execution of most procedures. Here we present four different approaches to non-terminal in vivo blood sampling techniques in either conscious or anaesthetized guinea pigs.

豚鼠非终端采血技术

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

从血液高效的iPS细胞生成使用附加体和HDAC抑制剂

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Detecting Establishment of Shared Blood Supply in Parabiotic Mice by Caudal Vein Glucose Injection

Parabiosis is an experimental method for surgically combining two parallel animals along the longitudinal axis of the body. We present a protocol for detecting the successful establishment of blood chimerism in parabionts by a caudal vein injection of glucose. Parabiotic mice were constructed. Glucose was injected into the donor mouse through the tail vein, and the fluctuation of blood glucose level was measured in both mice using a blood glucometer at different time points. Our results showed that after glucose injection, the blood glucose level in donor mice increased sharply after 1 min and decreased slowly thereafter. Meanwhile, the blood glucose level of the recipient mice peaked 15 min after injection. Similar results were obtained with Evans blue, used as a positive control for glucose. The synchronous fluctuation of blood glucose levels indicates that blood flow between the two mice was established successfully.

Here we describe a new method of detecting successful establishment of shared blood circulation of two parabionts through a caudal vein injection of glucose, which causes minimal damage and is not fatal to the parabionts.

考达尔静脉葡萄糖注射剂在益生菌小鼠中检测共享血液供应的建立

Parabiosis is an experimental method for surgically combining two parallel animals along the longitudinal axis of the body. We present a protocol for ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

一种从小鼠中分离大脑围取体的高输出方法

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...