授权号码P01 AI055637和NIAID / NIH U54 AI057156描述该项目的支持。其内容完全是作者的责任，并不一定代表办公室的RCE的程序，NIAID的，或美国国立卫生研究院的官方意见。

1天：F tularensis板接种<ol><li>板冻结的股票文化楼tularensis上的Mueller - Hinton琼脂辅以2.5％（体积/体积）捐助小牛血清，2％（体积/体积）IsoVitaleX，葡萄糖0.1％（WT /卷），和0.025％（WT /卷）铁焦。成长楼在37℃，5％的CO 2约24小时的 tularensis </li></ol>第2天：F tularensis液体介质接种<ol><li>准备和反应釜1公升阳离子调整穆勒韩丁中等（含1.23毫米二水氯化钙和1.03毫米的镁氯化）。当冷却到37℃，进一步补充液，葡萄糖0.1％（WT /卷），0.025％（WT /卷）焦磷酸铁，和2％（体积/体积）IsoVitaleX。过滤消毒（0.22μ）分为两个500毫升分装的介质。 </li><li>取出50毫升的无菌猎鹰管12毫升补充穆勒韩丁中期和转让。 </li><li>使用10μL无菌接种环，刮楼大loopful穆勒韩丁中等（见步骤2）到12毫升的琼脂（从第1天）和转让细菌 tularensis增长。液多次打破团块，并准备同质的菌悬液。不要旋涡。 </li><li>使用无菌吸管，接种5毫升菌悬液从第3步，每500毫升的容器。增长肉汤培养37℃，14日至18 h，轻轻摇动在新不伦瑞克省伊诺2300系列振动筛（190-200 RPM）。 </li></ol>第3天：Spheroplasting，渗透裂解，和蔗糖密度梯度离心<ol><li>获得F。从摇床tularensis文化和删除1毫升等分检查各自的光密度从每500毫升的文化。使用楼时，我们已经找到了最佳的膜萃取和隔离的结果tularensis年初增长0.2至0.4之间（〜10 7 10 8 CFU /毫升），这与OD 600相关的对数生长期的细胞。外径600小于0.2的文化就不会产生足够数量为后续的蔗糖梯度膜材料。相反，外径600比0.4（接近固定相）更大的文化已发现产生混合膜融合。 </li><li> 7500 X克文化离心30分钟，15 ° C至收集细胞。我们建议在4个250毫升的离心瓶离心文化，因为它有利于以后的颗粒悬浮。 </li><li>小心取出离心瓶从每个媒体上清和坚决自来水吸水材​​料的瓶子，以除去多余的生长介质。 </li><li>离心10 min内完成后，暂停在8.75毫升的蔗糖（5毫米的Tris，pH值7.5）0.75米，每个菌体。应暂停所有四个细菌颗粒（共35毫升菌悬液）10分钟内转移到无菌的250毫升的瓶（一个小stirbar）。 </li><li>在stirplate细胞悬液轻轻混匀，慢慢超过10分钟的过程中添加70毫升的10毫米乙二胺四乙酸二钠（2卷）（5毫米三，pH值7.8）。加入EDTA溶液与细胞悬液水平以下的移液器提示，以避免局部浓度升高的EDTA。 stirbar应旋转的速度足够充分混合细胞悬液，但速度还不够快，造成起泡或气泡的形成。已加入EDTA溶液后，孵育在室温下30分钟的解决方案。 </li><li> 30分钟潜伏期后，慢慢加入11毫升2毫克/毫升溶菌酶溶液的终浓度为200微克/毫升（1 / 10卷）。继续在溶菌酶的解决方案，除了混合细胞悬液。如上所述，添加下面的细胞悬浮液水平的枪头溶菌酶的解决方案，以避免局部浓度升高，溶菌酶。股票溶菌酶的解决方案（2毫克/毫升）可准备一次性使用的分装和储存在-20 ° C三至四个月。孵育在室温下30分钟的最终的解决方案。 </li><li>在30分钟的孵化，准备一个小stirbar室温Cellgro分子级的dh 2 O（4.5卷）和530毫升的无菌1L容量瓶中。 </li><li>渗透溶解缓慢稀释（流速约11毫升/分钟）的分子级DH 2 O（步骤7）在10至15分钟，轻轻搅拌成530毫升细胞悬液（步骤6） 。因为在这一步溶液的体积较大，增加stirbar的速度，以确保适当的混合。 stirbar应旋转的速度足以彻底驱散细胞悬液，一旦添加到DH 2 O，但不够快，导致泡沫或气泡的形成。加入烧瓶底部，毗邻的stirbar休息枪头的细胞悬液，以利统一的CEL稀释升停牌。我们发现，25毫升的吸液管工作在这一步中最好的。仔细监测稀释过程和中断必要分散的细胞，细胞团块，和细胞缕缕当地积累的流量。孵育在室温下30分钟的最终的解决方案。请注意，这一步看来是最具挑战性和实验的整体成功的关键之一。 </li><li>经过30分钟的孵化，转移到50毫升锥形管和离心渗透裂解液40毫升分装在30在10分钟XG 7500 ° C，移除了完整的细胞和碎片。 </li><li>离心后，小心地从每一个锥形管和池上清液在无菌1L容量瓶中27至30毫升上清液</li><li>转让25毫升，每池上清液，到16超速离心机管和离心机在200,000 X克（44400 RPM在F50L 8x39 FiberLite超速离心机转子）为2小时4 ° C。请注意，每个转子可容纳8管的，所以这一步需要两个转子和两个ultracentrifuges。另外，第二组8管应在4℃直至离心。 </li><li>超速离心机运行后10分钟内，准备修改膜再悬浮液。 8毫升膜悬浮液（25％[WT / WT]蔗糖，三5毫米，30毫米氯化镁2）转移到无菌管中，并添加1片EDTA无蛋白酶抑制剂的鸡尾酒和375 Benzonase ü。轻轻混合溶液溶解蛋白酶抑制剂片。 </li><li>经过离心，小心地倒出上清液，超速离心机管倒置在吸水材料，并坚决自来水管，除去多余的上清液。每个膜颗粒标记一个标记，以帮助在可视化的位置。改性膜再悬浮缓冲各600μL，轻轻重悬八个膜颗粒。每个膜再悬浮转移到下一组八管，轻轻悬浮在无菌管这八个小球，和游泳池膜resuspensions。颗粒膜是很难重悬，所以继续通过对颗粒的改性膜再悬浮缓冲，直到它果皮远离管壁。这是经常要使用微量提示，在再悬浮过程中的膜颗粒刮去管壁和援助。移液时，应避免泡沫或泡沫的形成。请注意，这一步看来是最具挑战性和实验的整体成功的关键之一。 </li><li>当膜颗粒已重悬，并从所有16个管拆除，600改性膜再悬浮缓冲液冲洗，每四个管子。应重点围绕标记的区域，每个管（膜颗粒）洗。转移到膜的再悬浮管清洗。其余的三四个管子套重复清洗步骤。 </li><li>孵育膜在室温降解减少絮状物质DNA和援助为30分钟的温和的摇摆再悬浮。 </li><li>删除膜悬挂小等份，并进行蛋白定量，以确定总膜产量。我们通常准备未稀释，1:1稀释（2.5％SDS）和1:3稀释（2.5％SDS）膜样品，加热样品在90 ° C为10分钟，用BioRad公司的DC蛋白测定量化膜再悬浮的蛋白浓度。总蛋白的产量一般在1.0 mg / ml和1.6毫克/毫升。热和SDS的使用要求释放使一个更准确的蛋白定量值可从提取的膜的外膜蛋白。 </li><li>准备到14 × 95毫米超清晰的，按下列顺序超速离心机管的线性蔗糖梯度分层蔗糖溶液1.8毫升（WT / WT，准备在5毫米EDTA，pH值7.5）：55％，50％，45％， 40％，35％，30％。蔗糖梯度分层是最简单的方法执行时，使用一个灯泡移液器，而不是自动移液器。 </li><li>基于膜再悬浮蛋白定量（16步），每个梯度顶部的膜再悬浮层1.5毫克。每个14 × 95毫米超速离心机管的最大容量为12.5毫升，从第17步的蔗糖溶液占10.8毫升，你不应该负载超过1.7毫升每梯度再悬浮膜。单独权衡和仔细调整每个蔗糖密度梯度管的最终重量（添加或删除膜再悬浮），使所有的管子的重量相同（± 0.01克）。 </li><li>转移到一个SW - 40Ti水平转头和离心蔗糖梯度256000 X克（38,000 RPM）至少17小时在4 ° C。 </li></ol>第4天：收集蔗糖梯度分数<ol><li>离心后17 h，停止离心机的运行，并仔细清除SW - 40Ti转子蔗糖梯度。 </li><li>仔细清洁用70％乙醇的蔗糖密度梯度管的底部，穿刺与21克针管的底部，并收集到无菌的离心管连续500μL分数梯度。重复随后的梯度。渐变应允许重力流滴，所以尽量不要去打扰蔗糖梯度。然而，如果梯度管的底部不再滴水，少量的压力可能会适用于使用的手指管。 </li><li>确定使用验光仪的分数每个蔗糖梯度折射率和折射率用 1克/毫升的密度相关。蛋白定位评估准备代表蔗糖梯度组分（密度为0.01增量由1.11克/毫升1.26克/毫升），进行SDS - PAGE分离，转移至硝酸纤维素和免疫印迹。 </li></ol>代表性的成果当正确执行，这一过程的结果，在IM和OM囊泡从两种类型的完全分离（如SchuS4）和B型（例如，LVS）株楼tularensis。代表免疫印迹显示在图1，F。 tularensis外膜蛋白本地化之间的密度为1.17和1.20克/毫升。相比之下，IMPS本地化之间的密度为1.13和1.14克/毫升。 <img src="/files/ftp_upload/2044/2044fig1.jpg" alt="图1" /> 图1免疫楼tularensis蔗糖密度梯度。从梯度和密度（克/毫升），根据折射率计算连续分数。蛋白经SDS - PAGE分离，转移到硝酸纤维素，和immunoblotted检测OMP和IMP的分馏。兆瓦，预染标准分子量大小（kDa）的左侧的每一个印迹指出。 LVS的，整个楼的细胞裂解液tularensis LVS的。相应的蔗糖梯度分数密度如上所述各自的车道。外膜蛋白和IMPS，在左旁指出，多克隆，单特异性抗血清检测。类似的结果，获得了楼tularensis SchuS4。

<ol>
	<li>Price, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrifugation+in+density+gradients.">Centrifugation in density gradients.</a> , 335-335 (1982).</li><li>Osborn, M. J., Gander, J. E., Parisi, E., Carson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+assembly+of+the+outer+membrane+of+Salmonella+typhimurium.+Isolation+and+characterization+of+cytoplasmic+and+outer+membrane.">Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane.</a> J Biol Chem. 247 (12), 3962-3962 (1972).</li><li>Nikaido, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+outer+membranes.">Isolation of outer membranes.</a> Methods Enzymol. 235, 225-225 (1994).</li><li>Mizuno, T., Kageyama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+characterization+of+the+outer+membrane+of+Pseudomonas+aeruginosa.">Separation and characterization of the outer membrane of Pseudomonas aeruginosa.</a> J Biochem. 84 (1), 179-179 (1978).</li><li>Huntley, J. F., Conley, P. G., Hagman, K. E., Norgard, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Francisella+tularensis+outer+membrane+proteins.">Characterization of Francisella tularensis outer membrane proteins.</a> J Bacteriol. 189, 561-561 (2007).</li><li>Huntley, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+outer+membrane+proteins+protect+mice+against+pulmonary+challenge+with+virulent+type+A+Francisella+tularensis.">Native outer membrane proteins protect mice against pulmonary challenge with virulent type A Francisella tularensis.</a> Infect Immun. 76 (8), 3664-3664 (2008).</li><li>Bevanger, L., Maeland, J. A., Naess, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agglutinins+and+antibodies+to+Francisella+tularensis+outer+membrane+antigens+in+the+early+diagnosis+of+disease+during+an+outbreak+of+tularemia.">Agglutinins and antibodies to Francisella tularensis outer membrane antigens in the early diagnosis of disease during an outbreak of tularemia.</a> J Clin Microbiol. 26 (3), 433-433 (1988).</li><li>Hubalek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+proteome+database+of+Francisella+tularensis.">Towards proteome database of Francisella tularensis.</a> J Chromatogr B Analyt Technol Biomed Life Sci. 787 (1), 149-149 (2003).</li><li>Pavkova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Francisella+tularensis+live+vaccine+strain:+proteomic+analysis+of+membrane+proteins+enriched+fraction.">Francisella tularensis live vaccine strain: proteomic analysis of membrane proteins enriched fraction.</a> Proteomics. 5 (9), 2460-2460 (2005).</li><li>Sandstrom, G., Tarnvik, A., Wolf-Watz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunospecific+T-lymphocyte+stimulation+by+membrane+proteins+from+Francisella+tularensis.">Immunospecific T-lymphocyte stimulation by membrane proteins from Francisella tularensis.</a> J Clin Microbiol. 25 (4), 641-641 (1987).</li><li>Sjostedt, A., Tarnvik, A., Sandstrom, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+T-cell-stimulating+17-kilodalton+protein+of+Francisella+tularensis+LVS+is+a+lipoprotein.">The T-cell-stimulating 17-kilodalton protein of Francisella tularensis LVS is a lipoprotein.</a> Infect Immun. 59 (9), 3163-3163 (1991).</li><li>Twine, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Francisella+tularensis+proteome:+low+levels+of+ASB-14+facilitate+the+visualization+of+membrane+proteins+in+total+protein+extracts.">Francisella tularensis proteome: low levels of ASB-14 facilitate the visualization of membrane proteins in total protein extracts.</a> J Proteome Res. 4 (5), 1848-1848 (2005).</li></ol>

这个协议描述一个spheroplasting，渗透裂解变化，和蔗糖密度梯度离心“金标准”的其他细胞成分2，3，4，革兰阴性细菌OMS的物理分离和浓缩。而我们前面描述的这种方法的应用，为隔离两种类型OMS A和B型株的楼tularensis 5，此演示，提供了奥姆隔离更详细和优化的过程。事实上，这是一个从这种致命的病原体识别潜在的表面暴露和推定致病因素的重要进展。在此过程中的意义是表明我们给予实质性的保护，对剧毒A型楼纯化的外膜蛋白，免疫小鼠的实验室研究tularensis肺挑战6。如上所述，细菌细胞，在渗透裂解仔细监测，和温柔的再悬浮膜颗粒的生长阶段出现的关联，这种膜分离过程中的成功/失败的关键步骤。虽然这种优化方法是严谨和一些实验性的变异，导致OMS相比，从使用的洗涤剂，氯化锂，钠碳酸盐7，8，9，10，11囊括出现显着提高纯度 ， 12。

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Donor calf serum</td>
<td>&nbsp;</td>
<td>Mediatech</td>
<td>35-022-CV</td>
<td>&nbsp;</td>
<tr>
<td>IsoVitaleX</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>211876</td>
<td>&nbsp;</td>
<tr>
<td>Cellgro molecular grade dH2O</td>
<td>&nbsp;</td>
<td>Mediatech</td>
<td>46-000-CM</td>
<td>&nbsp;</td>
<tr>
<td>38.5 ml polycarbonate bottle</td>
<td>&nbsp;</td>
<td>FiberLite</td>
<td>010-0514</td>
<td>&nbsp;</td>
<tr>
<td>F50L-8x39 ultracentrifuge rotor</td>
<td>&nbsp;</td>
<td>FiberLite</td>
<td>096-087051</td>
<td>&nbsp;</td>
<tr>
<td>Complete mini EDTA-free protease inhibitor cocktail</td>
<td>&nbsp;</td>
<td>Roche Diagnostics</td>
<td>04693132001</td>
<td>&nbsp;</td>
<tr>
<td>Benzonase</td>
<td>&nbsp;</td>
<td>Novagen</td>
<td>70664-3</td>
<td>&nbsp;</td>
<tr>
<td>DC protein assay</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>500-0116</td>
<td>&nbsp;</td>
<tr>
<td>Ultra-Clear 14x95mm centrifuge tubes</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>344060</td>
<td>&nbsp;</td>
<tr>
<td>SW-40Ti swinging bucket rotor</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>331301</td>
<td>&nbsp;</td>
<tr>
<td>Abbe benchtop refractometer</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>13-964-10 </td>
<td>&nbsp;</td>		</tbody>
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method for the isolation of francisella tularensis outer membranes

弗朗西斯tularensis是一种革兰氏阴性细胞内coccobacillus和人畜共患病兔热病的病原体。当与其他细菌病原体的感染剂量极低（&lt;10 CFU），疾病进展迅速，高发病率和死亡率表明， 弗朗西斯的毒株为新的致病因素的编码相比。暴露于表面的分子，即外膜蛋白（OMPS），已被证明是促进细菌宿主细胞结合，条目，细胞内生存，毒力和免疫逃避。进一步强调的事实，他们可以作为对一些细菌性疾病的保护疫苗研究外膜蛋白的相关性。鉴于可以从革兰氏阴性菌中提取的，通过批量膜萃取技术，包括超声离心和/或洗涤剂提取细胞的外膜蛋白，这些准备工作往往与周质和/或细胞质（内）膜（IM）污染物污染。多年来，为革兰氏阴性IM和外膜的生化和生物物理分离方法的“金标准”（OM），已受细菌spheroplasting和渗透裂解，蔗糖密度梯度离心。一旦蔗糖梯度层，OMS可以从IMS分离的基础上浮力密度的差异，被认为在很大程度上存在在OM脂多糖（LPS）的前提。在这里，我们描述了一个严谨和优化的方法来提取，丰富和隔离楼tularensis外膜和其相关的外膜蛋白。

Watch this Scientific Journal Video about Method for the Isolation of Francisella tularensis Outer Membranes at JoVE.com

Method for the Isolation of Francisella tularensis Outer Membranes

内层和外层膜分离议定书<em&gt;弗朗西斯tularensis</em&gt; spheroplasting，渗透溶解，和蔗糖密度梯度离心。

隔离方法弗朗西斯tularensis外膜

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other ...

method-for-the-isolation-of-francisella-tularensis-outer-membranes

Nanyang Technological University

Research

JoVE Journal

Biology

Method for the Isolation of Francisella tularensis Outer Membranes

17.0K 次观看.  University of Texas Southwestern Medical Center. 内层和外层膜分离议定书弗朗西斯tularensis spheroplasting，渗透溶解，和蔗糖密度梯度离心。

视频: Method for the Isolation of Francisella tularensis Outer Membranes

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

对小鼠胰腺胰岛分离和细胞内cAMP的测定方法

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

科研

生物学

Isolation of Neonatal Extrahepatic Cholangiocytes

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.
Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established1-4. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.
This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.

A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology.

新生儿肝外胆管的隔离

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

鼠冠状动脉血管平滑肌细胞的分离

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤:一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

一种快速，为隔离，功能可扩展的研究方法，以及细胞衍生的细胞外基质的分析

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

拟南芥种子高通量灭菌的标准化方法

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations.

荧光激活细胞分拣，用于分离瘦杆菌细胞群

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...