这项工作是支持由美国国家科学基金会（EAR-0920299，EAR-0745808），美国国家科学基金会东亚和太平洋夏季研究院，美国地质学会的研究资助计划和校友资助研究生研究奖学金补助从美国俄亥俄州立大学。我们要感谢编辑和两位匿名审稿人提出宝贵修改意见。

1。 MTB收藏<ol><li>决定在淡水网站收集趋磁细菌（MTB）时，它往往是最好的开始，一个池塘或滞销，有一个软泥质沉积物层流。在这个演示中，我们收集了样品Olentangy河的边缘，在校园里，在哥伦布市俄亥俄州立大学（OSU），俄亥俄州（美国）。虽然这是一个方便的位置，我们的演示，这里所描述的协议是适用于任何水生的位置。在该协议中所使用的材料，在表1中可以发现。查找位置的水的深度是10和100厘米之间。在这样的位置，你应该收集的最上层的沉积物用清晰的，螺旋盖的容器内。舀到容器中的沉淀物和水，直到它被填充有三分之一到二分之一的沉积物和与水的剩余量。保持容器淹没，直到它被装满了水，然后盖紧瓶盖的容器瓦特第i个其螺杆顶盖。这是没有必要的，混合的泥沙。擦拭外容器用毛巾擦干，然后把您的实验室样品。这是没有必要急于样品，您的实验室。我们已经离开MTB样品的前几天把他们带回我们的实验室在该领域的塑料容器。 ，MTB应该是可行的，为几个星期到几个月，只要为您储存在阴凉，阴凉的地方，在该领域的样品。 </li><li>一旦样品是在您的实验室，松开帽和离开它覆盖容器，以减少的蒸发量。存储容器，在室温下，在黑暗的房间，抽屉，或完全覆盖的容器用铝箔。允许的沉积物和细颗粒，以完全沉淀到容器的底部离开样品静置几个小时至几天。这是没有必要混合沉积物，MTB宁愿不受干扰的环境。清晰的塑料容器壁将让你确认，颗粒稳定的底部。 MTB可以根据您的样品，仍然活着，在许多个月的样本。 </li></ol> 2。 MTB隔离<ol><li>当您准备好隔离MTB，地点磁铁的塑料容器的两侧约1厘米以上的沉积物-水界面（ 图1A）。要小心，不要打扰在容器底部的沉积物。一侧上的相对侧上（ 图1A）的容器和另一个条形磁铁的北侧放置条形磁铁的南极。几乎任何磁铁都可以使用，例如一个磁性搅拌棒或大冰箱磁铁。可以使用任何支持的磁铁在上面的沉积物 - 水界面正确的高度。我们发现，休息的纸板或塑料盒的顶部上的磁铁是最好的，然而，磁体也可以贴在塑料容器的外部。等待30分钟到几个小时的细菌游泳的磁铁。 </li><li>用无菌吸管仔细收集水的容器内（ 图1A）位置附近的东南极的条形磁铁（在北半球收集的样本）。这水应该包含MTB都被吸引到南极条形磁铁。接着，毛细管赛道应被用来进一步丰富的MTB。 </li></ol> 3。山地车赛道<ol><li>为了丰富您的趋磁细菌的样品，毛细管的赛道是必要的（ 图1B和1C）。这些都需要在之前的透明塑料容器分离的细胞。 </li><li>使用5.75英寸（146毫米）的玻璃巴斯德吸管跑马场。使用金​​刚石笔或文件切断的移液管的顶部，吸移管的长度是不关键的，但它应该是能够包含约1-2毫升的水。接下来，使用本生灯融化的小费，所以它变得密封（ 图1C）。将得到的移液管，应具有一个开口端和一个密封端。 </li><li>请几个这些赛道，然后高压灭菌器。此外，您将需要进行高压灭菌棉和几个长的金属针。 </li><li>将过滤后的样品水，收集从图1A中所示的泥沙水界面附近，使用一个长的金属针连接到过滤注射器蒸压赛马场。过滤器的孔径大小应为0.22毫米至消除碎屑和杂质从水中。这是重要的，必须绝对确定，没有气泡的玻璃毛细管中。 </li><li>将底部的赛道上用无菌棉（ 图1B）。使用金属针推棉花朝向密封端的赛道，所以它是0.5 - 1 cm的距离，从密封的尖端（ 图1C）。 </li><li>使用无菌移液管，添加的MTB含水（从第2.2节）到样品ŕeservoir（开口），准备好的山地车赛道（ 图1B）。 </li></ol> 4。 MTB富集<ol><li>一旦在赛道充满了流体样品，奠定它在其一侧的水平表面上（ 例如 ，台式），并把条形磁铁的南极（在北半球）下一个密封的尖端赛车场（ 图1B和图1C）。 </li><li>等待5到30分钟的MTB迁移通过棉花。然后，你应该收集赛道上的尖端附近的流体。等待的时间太长，可以引入污染物，如其他的活动细菌，在毛细管的前端。或者，您也可以使用光学显微镜的赛道上，以查看提示和观看的的MTB收集在赛道上的尖端。这将允许您确定需要多长时间的山地迁移到棉塞。 </li><li>轻轻地用钻石刀做一点点划痕附近的棉塞和折断的赛道结束。 </li> &lt;李&gt;使用一个狭窄的针（规格为25或27）1毫升注射器从尖端的赛道上卸下流体。这种液体样品应含有丰富的MTB。 </li></ol> 5。 MTB通过光学显微镜观察<ol><li>将下降（10-20微升）的盖玻片上的丰富MTB样品。 </li><li>快速翻转盖玻片下拉朝下和盖玻片上挂着。 </li><li>盖玻片放置到放置在载玻片上的O形环。的O形环应具有稍小的直径然后盖玻片（大约1厘米， 图2）。 </li><li>把这个挂下降到光学显微镜阶段，集中在一个边缘的下降。一个60X干目标非常好，因为最具有较高的数值孔径（NA， 例如 ，0.93），但不需要油，这是很难用悬滴法（ 图2）。 </li><li>将酒吧磁铁靠近南端的悬挂ðROP和MTB将开始移向边缘最近的磁铁（ 图3）的下拉。在几分钟之内，许多MTB应的下拉式菜单中的边缘处（ 图3）。证明自己细菌通过扭转的磁铁的磁极是磁性的，然后在相反的方向观察细菌游泳。 </li></ol> 6。 MTB观察，用透射电子显微镜（TEM） <ol><li>放置到一个铜网的富集MTB下降（〜20微升），并允许在10分钟细菌定居。 </li><li>威克把多余的水用一块干净的滤纸。 </li><li>任选地，可以负染网格与2％乙酸双氧铀，2％磷钨酸的pH 7.2，或2.5％的钼酸钠7，8，9。这是通过放置在铜网格到一滴色斑立即孵育后的网格与富集MTB。孵育网格与负染色，时间将有所不同，这取决于在st上艾因使用，然后灯芯关闭流体用一块干净的滤纸。 </li><li>观察使用透射电子显微镜（TEM， 图4）的MTB。对于这里所描述的工作MTB吸附福尔瓦稳定和碳包覆200目铜网格（特德·佩拉＃01800）。在网格置于与碳侧向下下降的细胞悬浮液达10分钟，然后立即洗涤一次，由放置在网格上的一滴水，持续30秒。染色，电网下降2％的醋酸铀（特德·佩拉＃19481）30秒〜5分钟，然后完全干燥的滤纸采用了一块。通过TEM使用FEI TECNAI精神在80KV或FEI TECNAI F20的使用高角度环形暗场干200KV电网进行了分析。 </li></ol>

<ol>
	<li>Blakemore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetotactic+bacteria.">Magnetotactic bacteria.</a> Science. 190, 377-379 (1975).</li><li>Blakemore, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetotactic+bacteria.">Magnetotactic bacteria.</a> Annual Reviews in Microbiology. 36, 217-238 (1982).</li><li>Bazylinski, D. A., Frankel, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Biomineralization+of+Magnetite+(Fe3O4)+and+Greigite+(Fe3S4)+in+a+Magnetotactic+Bacterium.">Controlled Biomineralization of Magnetite (Fe3O4) and Greigite (Fe3S4) in a Magnetotactic Bacterium.</a> Applied and Environmental Microbiology. 61, 3232-3239 (1995).</li><li>Lefevre, C. T., Menguy, N., 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=A+Cultured+Greigite-Producing+Magnetotactic+Bacterium+in+a+Novel+Group+of+Sulfate-Reducing+Bacteria.">A Cultured Greigite-Producing Magnetotactic Bacterium in a Novel Group of Sulfate-Reducing Bacteria.</a> Science. 334, 1720-1723 (2011).</li><li>Simmons, S. L., Bazylinski, D. 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=South-seeking+magnetotactic+bacteria+in+the+Northern+Hemisphere.">South-seeking magnetotactic bacteria in the Northern Hemisphere.</a> Science. 311, 371-374 (2006).</li><li>Wolfe, R., Thauer, 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=A+'capillary+racetrack'+method+for+isolation+of+magnetotactic+bacteria.">A 'capillary racetrack' method for isolation of magnetotactic bacteria.</a> FEMS Microbiology Letters. 45, 31-35 (1987).</li><li>Rodgers, F. G., Blakemore, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+structure+in+a+many-celled+magnetotactic+prokaryote.">Intercellular structure in a many-celled magnetotactic prokaryote.</a> Archives of Microbiology. 154, 18-22 (1990).</li><li>Moench, T. T., Konetzka, W., 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=A+novel+method+for+the+isolation+and+study+of+a+magnetotactic+bacterium.">A novel method for the isolation and study of a magnetotactic bacterium.</a> Archives of Microbiology. 119, 203-212 (1978).</li><li>Balkwill, D., Maratea, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+of+a+magnetotactic+spirillum.">Ultrastructure of a magnetotactic spirillum.</a> Journal of Bacteriology. 141, 1399-1408 (1980).</li><li>Lins, U., Freitas, F., 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=Simple+homemade+apparatus+for+harvesting+uncultured+magnetotactic+microorganisms.">Simple homemade apparatus for harvesting uncultured magnetotactic microorganisms.</a> Brazilian Journal of Microbiology. 34, 111-116 (2003).</li><li>Jogler, C., Lin, W., 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=Toward+Cloning+of+the+Magnetotactic+Metagenome:+Identification+of+Magnetosome+Island+Gene+Clusters+in+Uncultivated+Magnetotactic+Bacteria+from+Different+Aquatic+Sediments.">Toward Cloning of the Magnetotactic Metagenome: Identification of Magnetosome Island Gene Clusters in Uncultivated Magnetotactic Bacteria from Different Aquatic Sediments.</a> Applied and Environmental Microbiology. 75, 3972-3979 (2009).</li><li>Lin, W., Li, 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=Newly+Isolated+but+Uncultivated+Magnetotactic+Bacterium+of+the+Phylum+Nitrospirae+from+Beijing,+China.">Newly Isolated but Uncultivated Magnetotactic Bacterium of the Phylum Nitrospirae from Beijing, China.</a> Applied and Environmental Microbiology. 78, 668-675 (2012).</li><li>Li, J., Pan, Y., 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=Biomineralization,+crystallography+and+magnetic+properties+of+bullet-shaped+magnetite+magnetosomes+in+giant+rod+magnetotactic+bacteria.">Biomineralization, crystallography and magnetic properties of bullet-shaped magnetite magnetosomes in giant rod magnetotactic bacteria.</a> Earth and Planetary Science Letters. 293, 368-376 (2010).</li><li>Oestreicher, Z., Valerde-Tercedor, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetosomes+and+magnetite+crystals+produced+by+magnetotactic+bacteria+as+resolved+by+atomic+force+microscopy+and+transmission+electron+microscopy.">Magnetosomes and magnetite crystals produced by magnetotactic bacteria as resolved by atomic force microscopy and transmission electron microscopy.</a> Micron. 43, 1331-1335 (2012).</li></ol>

没有利益冲突的声明。

趋磁细菌不一定在每一个水生环境，但是当他们这样做时，他们可以找到为100 - 1000细胞每毫升2。为了观察到的的MTB使用光学显微镜，你将需要大约50个细菌/ ml在您的样品8。如果没有或很少MTB在你的样品，然后你需要选择一个新的环境网站收集您的样品或你需要尝试的一个或多个在下一节中讨论的技术。 首先，你应该尝试从环境中收集更多的泥沙用一个大塑?...

<table border="1"><tbody><tr><td> 产品名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>载玻片</td><td> Fisher Scientific则</td><td> S95933 </td><td></td></tr><tr><td>玻璃巴斯德移液管</td><td> Fisher Scientific则</td><td> 13-678-6A </td><td></td></tr><tr><td> O-环</td><td>五金店</td><td></td><td></td></tr><tr><td>盖玻片</td><td> Fisher Scientific则</td><td> 12-542B </td><td></td></tr><tr><td>条形磁铁</td><td> Fisher Scientific则</td><td> S95957 </td><td></td></tr><tr><td>容器</td><td>任何</td><td></td><td>任何PLAST集成电路或玻璃容器中，可容纳至少为0.5 L和可以被密封</td></tr><tr><td>棉</td><td>任何</td><td></td><td></td></tr><tr><td>显微镜60X干镜头</td><td>蔡司</td><td></td><td> 60X干镜头是完全没有必要的，但不使用石油，这提供了非常高的NA </td></tr><tr><td>钻石笔</td><td> Fisher Scientific则</td><td> 08-675 </td><td></td></tr><tr><td> 0.22毫米滤光器</td><td> Fisher Scientific则</td><td> 09-719C </td><td></td></tr><tr><td> 1ml注射器</td><td> Fisher Scientific则</td><td> NC9788564 </td><td></td></tr><tr><td>微量离心管</td><td> Fisher Scientific则</td><td> 02-681-320 </td><td></td></tr><tr><td>福尔瓦/碳200目铜网上</td><td>特德·佩拉公司</td><td> 01800 </td><td></td></tr><tr><td>醋酸铀</td><td>特德·佩拉公司</td><td> 19481 </td><td></td></tr><tr><td> TECNAI精神TEM </td><td> FEI </td><td></td><td></td></tr><tr><td> TECNAI F20 S / TEM </td><td> FEI </td><td></td><td></td></tr></tbody></table>

collection, isolation and enrichment of naturally occurring magnetotactic bacteria from the environment

趋磁细菌（MTB）的水生微生物，特别是在美国马萨诸塞州的盐沼的沉积物样品于1975年1。自那时起已发现的MTB分层水和沉积物列来自世界各地的2。是，它们含有磁小，这是磁铁矿的细胞内，膜-结合的磁性纳米晶体铁（Fe 3 O 4）和/或胶黄铁矿铁（Fe 3 S 4），或两者3，4的一个共同特征的所有MTB。在北半球，MTB通常吸引到南端的一个条形磁铁，而在南半球，他们通常会吸引到北端的磁铁3,5。可以利用此属性时，试图从环境样品中分离出MTB。 丰富MTB的最常用的方法之一是使用一个透明的塑料容器收集沉淀物和水的天然来源，如一个淡水池。在北半球，南端的一个条形磁铁放在对外部的容器上面的泥沙沉积物 - 水界面。一段时间后，细菌可以从附近的容器的内部，用移液管的磁铁，然后通过使用毛细管赛马场6和磁体进一步丰富。一旦丰富，细菌可以被置于显微镜载片上，使用悬滴法，并在光学显微镜下观察到的，或沉积到铜网格，使用透射型电子显微镜（TEM）观测。 使用这种方法，被隔离MTB可以研究微观特性来确定，例如游泳行为，鞭毛，细胞形态的细胞，磁性的结晶形状，磁数，在每个单元格中的磁链数，组合物的类型和数量的的nanomineral结晶体，与细胞内空泡的存在。

一块磁铁是一种有效的工具，它可以被用来隔离磁细菌（MTB）的环境样品中（ 图1A）所载。的毛细管赛马场（ 图1B）使用的磁特性的MTB吸引他们通过棉塞在那里他们可以从非趋磁也包含在环境样品的微生物分离。 <img alt="图1" src="/files/ftp_upload/50123/50123fig1.jpg" /> 图1。一个透明的塑料瓶，含泥沙和水样采集?...

Watch this Scientific Journal Video about Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment at JoVE.com

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

我们展示了一个方法，可以应用到天然水域中收集的趋磁细菌（MTB）。 MTB可以从沉积物样品，用一个相对简单的设置，利用细菌的天然磁分离和富集。隔离MTB然后可以同时使用光学和电子显微镜详细检查。

从环境中自然产生的趋磁细菌的采集，分离和富集

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

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Biology

22.9K 次观看.  The Ohio State University. 我们展示了一个方法，可以应用到天然水域中收集的趋磁细菌（MTB）。 MTB可以从沉积物样品，用一个相对简单的设置，利用细菌的天然磁分离和富集。隔离MTB然后可以同时使用光学和电子显微镜详细检查。

视频: Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

NK细胞与干细胞技术的RosetteSep套件的人体血液的富集

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

科研

生物学

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 ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

小非编码RNA从人血清中分离

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. Birds are widely used subjects in biological research and are ideal for studies involving repeated sperm samples. However, few resources are currently available for those wishing to learn how to collect and extract DNA from avian sperm. Here we describe cloacal massage, a gentle, non-invasive manual technique for collecting avian sperm. Although this technique is established in the literature, it can be difficult to learn from the available descriptions. We also provide information for extracting DNA from avian semen using a commercial extraction kit with modifications. Cloacal massage can be easily used on any small- to medium-sized male bird in reproductive condition. Following collection, the semen can be used immediately for motility assays, or frozen for DNA extraction following the protocol described herein. This extraction protocol was refined for avian sperm and has been successfully used on samples collected from several passerine species (Passer domesticus, Spizella passerina, Haemorhous mexicanus, and Turdus migratorius) and one columbid (Columba livia).

This protocol describes cloacal massage, a non-invasive manual technique for collecting semen from birds, and DNA extraction from avian sperm using a commercial extraction kit with modifications.

泄殖腔按摩与精子 DNA 分离法采集禽精

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. ...

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ability to biomineralize magnetosomes, which are magnetic nanometer-sized crystals of magnetite (Fe3O4) or greigite (Fe3S4) surrounded by a lipid bilayer membrane, within their cytoplasm. For most known magnetotactic bacteria, magnetosomes are assembled in chains inside the cytoplasm, thereby conferring a permanent magnetic dipole moment to the cells and causing them to align passively with external magnetic fields. Because of these specific features, magnetotactic bacteria have a great potential for commercial and medical applications. However, most species are microaerophilic and have specific O2 concentration requirements, making them more difficult to grow routinely than many other bacteria such as Escherichia coli. Here we present detailed protocols for growing three of the most widely studied strains of magnetotactic bacteria, all belonging to the genus Magnetospirillum. These methods allow for precise control of the O2 concentration made available to the bacteria, in order to ensure that they grow normally and synthesize magnetosomes. Growing magnetotactic bacteria for further studies using these procedures does not require the experimentalist to be an expert in microbiology. The general methods presented in this article may also be used to isolate and culture other magnetotactic bacteria, although it is likely that growth media chemical composition will need to be modified.

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

We present a procedure for growing several strains of Magnetospirillum in two different types of growth media. Magnetospirillum gryphiswaldense strain MSR-1 is grown in both liquid and O2 concentration gradient semi-solid media while M. magneticum strain AMB-1 and M. magnetotacticum strain MS-1 are grown in liquid medium.

螺属的生长趋磁菌: 菌株 MSR-1、AMB-1 和 MS-1

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

成年小鼠泪液和下颌腺肌上皮细胞的分离

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

分离和富集人肺上皮祖细胞用于类器官培养

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.

城市环境 -环境水库SARS-CoV-2采样和检测管道

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

从真核细胞 中快速体内 固定和分离转化复合物

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

从人骨关节炎膝关节收集和RNA提取

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...