支持原始的上述研究是由美国国家科学基金会资助0344630。

鲎有关的出血的解剖特点（图1） <img src="files/ftp_upload/old/958_2008_10_3_0/958_Figure-01.png" alt="图1" width="389" height="259" /> 图1 <ol><li>人体的三个主要业务部门，由前到后，prosoma（P）opisthosoma（海外），和尾节（T）1。 </li><li> prosoma前和横向的自由空间的法兰。 </li><li> opisthosoma后最压痕，尾节阐述，是终端湾。 </li><li>联合prosoma的opisthosoma口齿铰链（H）。 </li><li>心脏是位于沿背中线，下方的甲壳prostoma和opisthosoma 2（图2）。 <img src="files/ftp_upload/old/958_2008_10_3_1/958_Figure-02.png" alt="图2" width="329" height="414" /> 图2 </li></ol>总的考虑，不育和保护暴露于脂多糖（内毒素） <ol><li>成功出血的主要敌人是细胞聚集，胞外分泌，coagulin血块的形成。细胞聚集和胞外分泌刺激脂多糖（又名，内毒素，LPS），革兰氏阴性菌的产物。洗涤细胞胞吐的LPS的阈浓度为0.1 - 1毫克/毫升，但悬浮在血浆中的细胞被激活显着降低浓度3。内毒素是由简单的高压灭菌不灭活，可以被假定为在表面上，并在解决方案和未认证的内毒素试剂。 </li><li>为了减少在收集血液凝血的机会，选择只有完美的，完好的动物出血。预冷却在4 ° C室温1-2 h的动物。实践无菌技术。避免与内毒素污染。 </li><li> LPS无3％的盐水在中医临床中使用，可以从医疗供应商购买（见专门的试剂和耗材表）。 LPS的潜伏期可去除玻璃器皿和金属在180 ° C，4小时。无菌一次性使用的注射器针头式的Petri培养皿，和螺丝帽的离心管都脂多糖，可以不修改，只要实行适当的无菌技术是使用。 </li><li>不同动物的血细胞的稳定性不同，与一些动物个体进行自发脱粒的细胞，导致coagulin血块的形成，即使动物是最大的照顾流血。其他个人鲎血细胞明显更稳定，并遵循适当的程序时，仍然在出血和细胞从血浆中分离完全颗粒状。 </li><li>据报道，几家代理商稳定的血细胞，下面的出血，包括咖啡因（脂多糖3％氯化钠为0.25卷为10 mM咖啡因流血）4，普萘洛尔（终浓度为1毫米）5 6，二甲基亚砜，二价阳离子螯合（葡萄糖为0.1米，0.03米的柠檬酸钠，柠檬酸0.026中号10毫米二钠乙二胺四乙酸（钠2 EDTA）的，pH值4.6）7，G蛋白和磷脂酶- C的途径的抑制剂（霍乱和同等体积的渗入百日咳毒素，U73122）8，氯替代磺酸钠阴离子9，氯离子通道阻滞剂9，9环腺苷拮抗剂，巯基试剂（5毫米N -乙基马来酰亚胺，NEM）5，和膜活性清洁剂吐温20（渗入到0.1卷2％Tween - 20的LPS无0.5 M氯化钠）。 </li></ol>用品和出血鲎试剂：收集大量的血液<ol><li>一个或多个大，完好的马蹄蟹</li><li>喷瓶含70％乙醇</li><li> Kimwipes </li><li> 14号针</li><li>坚固镊子</li><li>用冰块的冰桶</li><li> 50毫升的螺丝帽塑料一次性离心管</li><li>机架容纳50 mL管</li><li>反离心机</li><li>无菌50毫升血清移液器</li><li>灯泡贯流式吸液管填料</li><li>一次性使用无菌过滤设备</li><li>真空泵</li><li> LPS免3％NaCl溶液（血细胞的集合） </li><li>吐温20（血细胞的集合） </li></ol>大批量出血的筹备工作<ol><li>出血，使用大型的，完好的动物。冷藏冷房前出血1小时的动物。 </li><li>建立一个稳定的支持与冰桶，高约0.3米（邮寄冷水或冷冻试剂，一次性泡沫塑料海运集装箱适当）和经营者的低椅子的舒适安排。冰桶放在支撑结构和绘制椅子到本机。你应该在你的胳膊肘你的大腿上休息的椅子上长时间舒适的客厅，并用左手拿着一个苦苦挣扎的鲎血液收集管在冰桶冰床直立撑起以上定位。 </li><li>前寒意4 - 6螺丝封在冰浴50毫升的一次性塑料离心管。直立管周围的冰浴容器的周边范围的冰。前开始出血操作，删除从管和相邻的表格盖瓶盖，坚持无菌技术。 </li><li>删除第一个14 GA针和使用坚固镊子松开针在其袖子，但不删除针从袖子。保持无菌。切勿用手指触摸进针的任何部分。针针裸露的屁股与相邻的柜台上放置与套筒接触任何表面，以保持其不育性升高。 </li><li>一块与70％的乙醇Kimwipe。 </li></ol> 出血的动物（特别是右手操作） <ol><li>动物出血：prosoma前法兰和抓基地的尾巴（终端湾）的opisthosma后部分拇指接触的四个手指在左手抓住动物。腹面的动物面临的手掌。尾节在于沿在手腕的拇指和项目的基础。 Flex的动物轻轻prosoma和opisthosoma直角（图3a）。 <img src="files/ftp_upload/old/958_2008_10_3_2/958_Figure-03.png" alt="图3" width="360" height="360" /> 图3 </li><li>清洁外露的铰链关节连接prosoma用70％乙醇和opisthosoma应用乙醇蘸Kimwipe（图3b）。 </li><li>旋转的左手手指2 - 4至上，动物的背表面朝上。用右手，插入注射器针头14 GA，仍然在其保护套，左手手指2和3之间。拿镊子的右手，并删除其保护套针抓钳（图3c）的对接。左手旋转，使手的背水面面对你的左边，拇指和食指，和动物是其面临的右手背表面倒挂。定位于50 ml收集管和东方的动物，使针的尖底是在铰链关节，使针轴是平行的背中线（图3d）指出以上的屁股针。通过铰接插入针的尖底，入心腔，保持背中线平行针定位，并在中线与对接仍高于开放收集管定位针插入。针插入约1 - 2厘米深的prosoma的核心部分。只要心脏被刺破，血液会流向迅速（图4a，箭头），所以重要的是要确保注射器针头结束分娩是在收集管口的位置，收集的血液的初始浪涌。 </li><li>血液会退出的心，最初在一个连续的流，后约30 - 50毫升血液已收集，将放缓至个别滴（图4b）。为了改善血液流动，它可能是好的政策，重新定位针略尖。针倾斜向上或向左或向右滑动其尖端深入入心，或拉铰接找到位置，优化血液流动。不要过度压缩的动物，损害动物，因为这个风险，可能会造成淤血启动凝血。 <img src="files/ftp_upload/old/958_2008_10_3_3/958_Figure-04.png" alt="图4" width="360" height="360" /> 图4 </li><li>由于每个收集管与血液填充，旋转冰浴，使未来的空管，下针的结束分娩。 </li><li>如出血减慢，运行在逆行方向进针，入心有空气流动的风险。可能发生这种情况时，动物理顺，从而降低血压。尽量避免动物保持在弯曲的位置，或允许整顿仅出现缓慢。 </li><li>当动物捐血站，删除从心针出血。重新收集管帽，并立即离心机表离心机，1000转，5分钟。血细胞会形成紧凑颗粒的收集管底部。由于凝血的所有组件内的血细胞分泌囊泡中，尽快血浆是从细胞分离，血浆凝血的风险被淘汰。 </li><li>这是必须避免凝血瘀血。采取以下措施减少凝血的风险：预冷的动物，并保持在冰浴的收集管;柔和处理的动物;减少血液流动的速度与注射器针头，血液离心立即经过收集，避免污染的针头和收集管内毒素;维持无菌程序。正如上面所提到的的，一些动物个体，尤其是烦躁的血细胞，启动胞外分泌，即使采取了这些预防措施是其次。 </li><li>离心后，检查血液凝固的迹象管 - 细胞和凝胶状血块材料链连接管的墙壁，血块顶部的凝胶状物质，或者更糟的是，大量的血块包埋在血细胞液柱（图4c，箭头）。从这些管子的材料是血清，血浆，并含有血细胞分泌的产品。 </li><li>血浆转移到无菌试管中，用50 mL无菌血清吸管。移液时要小心，以避免吸细胞沉淀在试管底部的细胞。最好是离开以上的颗粒几毫升的血浆，以避免干扰细胞沉淀。 </li><li>上述步骤的目的是收集大量的血细胞分泌的产品未受污染的血浆卷。要收集大量的洗血细胞，血液收集到0.1 2％Tween - 20的LPS无3％NaCl溶液中溶解的卷。离心10分钟，轻轻的细胞，建立一个松散的细胞沉淀，然后用脂多糖3％氯化钠的几个变化的细胞。可以编写包含的血细胞的分泌产物的细胞提取LPS无蒸馏水洗涤细胞裂解。这裂解包含丰富的蛋白质和多肽的抗微生物免疫防御10,11，还含有coagulogen，酶原形式存在的结构蛋白外血块，并收集蛋白酶转换coagulogen到coagulin ，形成蛋白聚合成纤维血块12。这种提取物是类似市售的产品，amebocyte裂解或鲎鲎，这是用于检测存在脂多糖 13 。负责coagulogen水解修改coagulin蛋白酶级联反应被激活由LPS 14 。 </li><li>动物的处置后出血。出血上述方法进行动物耐受性好。在伍兹霍尔海洋生物实验室工作时，我返回后出血与期望，他们将生存的海洋动物。如果动物是从遥远的马蹄蟹的正常范围保持在水族箱，他们可以流血反复每月一次或两次。正如下文所述，维持在圈养的动物都需要高品质的水，并经常摄食，以保持他们的健康。安乐死可以通过破坏背侧神经节，这是坐落在两眼之间的背中线。 </li></ol>等离子处理和存储<ol><li>从蛋白酶的保护。如果血浆用于蛋白质纯化，它应与蛋白酶抑制剂治疗后，立即集合。加入phenylmethylsulfonylfluoride（PMSF）终浓度为1毫米。 PMSF保持为0.1 M的股票在DMSO或乙醇溶液。这是很不稳定的水 15，因此不能依靠持久的保护，但除了将灭活后立即分离血浆，血细胞可能已经从细胞中释放的蛋白酶的小批量。 </li><li>无菌过滤：超滤下使用一次性介质过滤装置，装有0.22毫米的孔隙​​过滤器真空等离子体消毒。这些过滤器堵塞的倾向。离心过滤，以减少这个问题之前，最好是等离子。如果大量的血浆处理，这是明智的预过滤血浆首先使用1毫米孔径一个微孔过滤器，然后用0.45毫米孔径微孔过滤。 </li><li> Plasma可以得到保护，免受细菌污染加入NaN的3至终浓度为0.2毫克/毫升，或无菌过滤。未消毒的血浆不应该超过2天，期间无一或正在采取的这些抗菌措施的其他存储。 </li><li>血液凝固。与哺乳动物的凝血系统，鲎血浆中含有凝血元素没有。相反，整个机械凝块的形成，血块的结构蛋白（coagulogen）和蛋白酶系统的过程coagulogen以使其能够聚合成不溶性凝块的，是发现在 16的血细胞的分泌颗粒。因此，如果被删除之前，他们有机会进行胞吐的血细胞，血浆凝血蛋白质是完全免费和将无限期地保持液体。 </li><li>蛋白质纯化。血蓝蛋白是目前在40毫克/毫升血浆中，是一个70 kDa的亚基48 MER。它可以隔离超速离心（40,000 X克，8小时），具有较大的孔隙大小，如排除BioGel A5m 17树脂或凝胶过滤。 C -反应蛋白存在于1 - 5毫克/毫升，并用钙螯合剂 18洗脱可以隔离phosphorylethanolamine琼脂糖亲和隔离。广谱蛋白酶抑制剂，2 macroglogulin，已纯化Sephacryl S - 300型树脂后血蓝蛋白和C -反应蛋白已被删除19凝胶过滤。 </li></ol> 文化运动型血细胞镜检 <ol><li> amebocyte草坪，在体外建立：LPS无3％NaCl溶液中添加无菌塑料的Petri式培养皿为35毫米的菜，为60毫米的菜2.5毫升，6毫升90毫升菜（1毫升）。血液是由心脏穿刺，使用上面介绍的方法获得，但与修改了23号，在无菌注射器针头1，而不是用14号针头。血液流动下拉下降，从23号针头收集到35毫米盘，3到60毫米盘滴，和100毫米盘9成滴1滴。血细胞，然后温柔纷飞，首先在一个圆形图案，然后再返回，来回在3％NaCl溶液混合均匀。准备5分钟，在室温T孵育使血细胞附着到培养皿的表面，然后选择3缓冲区最初的生理盐水取代。 </li><li>如果想要保持​​在一个undegranulated条件下的底层连接的血细胞，取代最初的生理盐水血浆混合新鲜脂多糖免费的3％氯化钠。等离子加速，甚至没有在LPS的自发的血细胞脱颗粒，其去除，稳定细胞undegranulated状态。 </li><li>如果最初的生理盐水取代用无菌血浆，血细胞转化的循环血细胞（图5）卵形的形状和扁平到基质，然后启动脱粒和一层coagulin外血块的形成以上夷为平地amebocytes的草坪 20 。 <img src="files/ftp_upload/old/958_2008_10_3_4/958_Figure-05.png" alt="图5" width="300" height="201" /> 图5 </li><li>建立汇总amebocytes植文化。当血液收集无菌，无脂多糖条件下，血细胞落户暂停和聚合，形成一个组织样肿块。一个方便的容器是玻璃胚盘呈现在180 0℃孵育4小时的LPS无使用19号针头穿刺心脏的血液收集在这个容器中的孵育1 - 在室温T，然后用18号无菌注射器针头amebocyte总量削减1平方毫米的片2小时，夹在两coverglasses或玻璃罩和幻灯片coverglasses碎片分离出来，作为垫片。约半小时后，amebocytes开始从外植体的外围迁移到盖玻片（图6A），在那里他们可以观看相衬或 21 DIC的光学。起初，细胞迁移是透明伪足（图6b（M））以上的移民底层扩展紧凑，和，然后降低接触表面和提出的议案涉及的颗粒状细胞质流动到伪足22 。后来，细胞扁平化（图6B（F）），并开始脱颗粒（图6b（D）），并停止运动 23 。这里所描述的文化室，让不同的实验药物灌注到文化室，以探讨其对细胞运动和颗粒胞吐9的影响。延长amebocyte文化的方法论审查镜检可以发现在 24 。 <img src="files/ftp_upload/old/958_2008_10_3_5/958_Figure-06.png" alt="图6" width="480" height="327" /> 图6 </li></ol>成年马蹄蟹在水族箱维护马蹄蟹需要高品质的海水，可保持在装有水净化系统，可提供微粒过滤，曝气，脱硝水 25的水族箱。虾，龙虾，鱼，鱿鱼或动物应该是吃一个星期2或3倍。进料是通过去除动物的水族馆，解放军清在它的后面，并放在嘴里的食物。如果饿了，动物不久将开始吞咽食物。根据海水系统的复杂程度，动物应保持一个小时左右，直到它排粪在海水中的一个单独的容器或已经开始喂养后，它可以返回到水族馆。长期维护，动物应保持远离光线，以防止甲壳表面绿色或蓝绿色藻类的生长。藻类侵蚀的甲壳和自成年不蜕皮，这将导致死亡的动物26。在野生动物部分埋在沙的时间花费不多，但在水族箱中的沙或碎石呈现在保持长期维护的清洁水平的重要的难度。

<ol>
	<li>Lochhead, J. H., Brown, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selected+Invertebrate+Types.">Selected Invertebrate Types.</a> , 360-381 (1950).</li><li>Shuster, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circulatory+system+and+blood+of+the+horseshoe+crab+Limulus+polyphemus+L.:+a+review.">The circulatory system and blood of the horseshoe crab Limulus polyphemus L.: a review.</a> US Dept Energy, Fed Regul. Comm. , (1978).</li><li>Conrad, M. L., Pardy, R. L., Wainwright, N., Child, A., Armstrong, P. 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L., Cohen, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+Cells+of+Marine+Invertebrates:+Experimental+Systems+in+Cell+Biology+and+Comparative+Physiology.">Blood Cells of Marine Invertebrates: Experimental Systems in Cell Biology and Comparative Physiology.</a> , 259-260 (1985).</li><li>Liang, S. M., Liu, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+Limulus+coagulation+system:+inhibition+of+activation+of+the+proclotting+enzyme,+by+dimethyl+sulfoxide.">Studies on the Limulus coagulation system: inhibition of activation of the proclotting enzyme, by dimethyl sulfoxide.</a> Biochem. Biophys. Res. Commun. 105, 553-559 (1982).</li><li>Soderhall, K., Smith, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+the+haemocyte+populations+of+Carcinus+maenas+and+other+marine+decapods,+and+prophenoloxidase+distribution.">Separation of the haemocyte populations of Carcinus maenas and other marine decapods, and prophenoloxidase distribution.</a> Dev. Comp Immunol. 7, 229-239 (1983).</li><li>Solon, E., Gupta, A. P., Gaugler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+during+exocytosis+in+Limulus+polyphemus+granulocytes.">Signal transduction during exocytosis in Limulus polyphemus granulocytes.</a> Dev. Comp. Immunol. 20, 307-321 (1996).</li><li>Armstrong, P. B., Rickles, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotoxin-induced+degranulation+of+the+Limulus+amebocyte.">Endotoxin-induced degranulation of the Limulus amebocyte.</a> Exp. Cell Res. 140, 15-24 (1982).</li><li>Iwanaga, S., Kawabata, S., Shuster, C. N., Barlow, R. B., Berkman, L., Brockmann, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+phylogeny+of+defense+molecules+associated+with+innate+immunity+in+horseshoe+crab.">Evolution and phylogeny of defense molecules associated with innate immunity in horseshoe crab.</a> Front Biosci. 3, (1998).</li><li>Armstrong, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+American+Horseshoe+Crab.">The American Horseshoe Crab.</a> , 288-309 (2003).</li><li>Iwanaga, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+limulus+clotting+reaction.">The limulus clotting reaction.</a> Curr. Opin. Immunol. 5, 74-82 (1993).</li><li>Levin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Limulus+amebocyte+lysate+test:+perspectives+and+problems.">The Limulus amebocyte lysate test: perspectives and problems.</a> Prog. Clin. Biol. Res. 231, 1-23 (1987).</li><li>Koshiba, T., Hashii, T., Kawabata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+structural+perspective+on+the+interaction+between+lipopolysaccharide+and+factor+C,+a+receptor+involved+in+recognition+of+Gram-negative+bacteria.">A structural perspective on the interaction between lipopolysaccharide and factor C, a receptor involved in recognition of Gram-negative bacteria.</a> J. Biol. Chem. 282, 3962-3967 (2007).</li><li>James, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+protease+inhibitor+phenylmethylsulfonyl+fluoride+in+buffers.">Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers.</a> Anal. Biochem. 86, 574-579 (1978).</li><li>Murer, E. H., Levin, J., Holme, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+studies+of+the+granules+of+the+amebocytes+of+Limulus+polyphemus,+the+horseshoe+crab.">Isolation and studies of the granules of the amebocytes of Limulus polyphemus, the horseshoe crab.</a> J. Cell Physiol. 86, 533-542 (1975).</li><li>Decker, H., Ryan, M., Jaenicke, E., Terwilliger, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SDS+induced+phenoloxidase+activity+of+hemocyanins+from+Limulus+polyphemus,+Eurypelma+californicum+and+cancer.">SDS induced phenoloxidase activity of hemocyanins from Limulus polyphemus, Eurypelma californicum and cancer.</a> , 276-17796 (2001).</li><li>Armstrong, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cytolytic+function+for+a+sialic+acid-binding+lectin+that+is+a+member+of+the+pentraxin+family+of+proteins.">A cytolytic function for a sialic acid-binding lectin that is a member of the pentraxin family of proteins.</a> J. Biol. Chem. 271, 14717-14721 (1996).</li><li>Armstrong, P. B., Melchior, R., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humoral+immunity+in+long-lived+arthropods.">Humoral immunity in long-lived arthropods.</a> J. Insect Physiol. 42, 53-64 (1996).</li><li>Armstrong, P. B., Armstrong, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+decorated+clot:+Binding+of+agents+of+the+innate+immune+system+to+the+fibrils+of+the+limulus+blood+clot.">The decorated clot: Binding of agents of the innate immune system to the fibrils of the limulus blood clot.</a> Biol. Bull. 205, 201-203 (2003).</li><li>Armstrong, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+the+motile+blood+cells+of+the+horseshoe+crab,+Limulus.+Studies+on+contact+paralysis+of+pseudopodial+activity+and+cellular+overlapping+in+vitro.">Interaction of the motile blood cells of the horseshoe crab, Limulus. Studies on contact paralysis of pseudopodial activity and cellular overlapping in vitro.</a> Exp. Cell Res. 107, 127-138 (1977).</li><li>Armstrong, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+of+the+Limulus+blood+cell.">Motility of the Limulus blood cell.</a> J. Cell Sci. 37, 169-180 (1979).</li><li>Armstrong, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+and+spreading+of+Limulus+blood+cells+on+artificial+surfaces.">Adhesion and spreading of Limulus blood cells on artificial surfaces.</a> J. Cell Sci. 44, 243-262 (1980).</li><li>Armstrong, P. B., Cohen, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+Cells+of+Marine+Invertebrates.">Blood Cells of Marine Invertebrates.</a> Lab. Animal. , 253-258 (1985).</li><li>Smith, S. A., Berkson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+culture+and+maintenance+of+the+horseshoe+crab+(Limulus+polyphemus).">Laboratory culture and maintenance of the horseshoe crab (Limulus polyphemus).</a> Lab. Animal. 34, 27-34 (1980).</li><li>Leibovitz, L., Lewbart, G. A., Shuster, C. N., Barlow, R. B., Brockmann, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+American+Horseshoe+Crab.">The American Horseshoe Crab.</a> , 245-275 (2003).</li><li>Rudkin, D. M., Young, G. A., Nowlan, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oldest+horseshoe+crab:+a+new+xiphosurid+from+Late+Ordovician+Konservat-Lagerstatten+deposits.">The oldest horseshoe crab: a new xiphosurid from Late Ordovician Konservat-Lagerstatten deposits.</a> Palaeontology. 51, 1-9 (2008).</li><li>Shuster, C. N., Sekiguchi, K., Shuster, C. N., Barlow, R. B., Brockmann, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The American Horseshoe Crab. , 103-132 (2003).</li><li>Walls, E. A., Berkam, J., Smith, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+horseshoe+crab,+Limulus+polyphemus,+200+million+years+of+existence,+100+years+of+study.">The horseshoe crab, Limulus polyphemus, 200 million years of existence, 100 years of study.</a> Rev. Fisheries Sci. 10, 39-73 .</li><li>Patten, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Evolution+of+the+Vertebrates+and+Their+Kin.">The Evolution of the Vertebrates and Their Kin.</a> P. Blakiston's Son and Co. , 195-209 (1912).</li></ol>

有4种鲎， 鲎波吕斐摩斯从北美东海岸和三个品种，范围从日本到孟加 ​​拉湾。动物被确定为“活化石”，因为解剖学上的类似形式已为化石发现4.45亿年前27。鲎代表一个长寿命的动物，需要10 - 13岁达到成熟和生活至少20年的 28 。虽然一直受到广泛的收获作为诱饵的鳗鱼，海螺渔业29，美国的鲎仍然相当丰富，并允许50毫升血从一个小的成人非破坏性的收集和，从高达400毫升大?...

blood collection from the american horseshoe crab, limulus polyphemus

鲎的任何最佳的长寿命无脊椎动物的免疫系统。马蹄蟹的免疫研究提供了便利，便于收集大量的血液，并从简单的血液。马蹄蟹只有在一般的流通，颗粒amebocyte一个单一的细胞类型。血浆海水中的盐含量，只有三个丰富的蛋白质，血蓝蛋白，呼吸蛋白，C -反应蛋白，在外来细胞的杀伤破坏的功能，包括细菌细胞和α2-巨球蛋白，能抑制蛋白酶入侵的病原体。血液收集的条件下，心脏穿刺直接脂多糖（又名，内毒素，LPS），革兰氏阴性菌的产品，最大限度地减少污染。大型动物可以产生200 - 400毫升的血液。等离子体的研究，立即清除血细胞从血浆离心的血浆，然后可以分割成它的组成蛋白质。方便的血细胞是研究微观LPS无等渗盐水（0.5 M氯化钠）的条件下，允许直接镜检培养皿的表面上放置一个多脂多糖免费coverglasses通过收集小体积血，然后安装这些coverglasses在简单的观察室，下面的细胞附着。一个直接观察的第二准备，以收集3 - 5毫升血液，在LPS无胚盘，然后explanting片段的聚合amebocytes一腔三明治组织之间的幻灯片和一个玻璃罩。在这个准备，运动型amebocytes迁移到盖玻片表面，在那里他们可以很容易地观察到。凝血系统涉及amebocytes聚集和形成血块，这是由血细胞分泌颗粒释放的一种蛋白质，coagulin外。洗血细胞的生化分析的需要，聚集和脱颗粒不会发生，它可以通过收集到0.1 2％的Tween - 20，0.5 M的LPS无盐，其次是细胞离心和洗涤用0.5的中号的卷的血液完成氯化钠。

Watch this Scientific Journal Video about Blood Collection from the American Horseshoe Crab, Limulus Polyphemus at JoVE.com

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

美国鲎，鲎波吕斐摩斯，无疑是最方便的任何无脊椎动物的血液大量源。血液成分简单，只有一个循环，颗粒amebocyte，只有三个蛋白质丰富的血浆，血蓝蛋白，C -反应蛋白和α2-巨球蛋白的细胞类型。发自内心的血液收集和离心分离血细胞和血浆。

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 ...

blood-collection-from-the-american-horseshoe-crab-limulus-polyphemus

Research

JoVE Journal

Biology

29.9K 次观看.  University of California, Davis. 美国鲎，鲎波吕斐摩斯，无疑是最方便的任何无脊椎动物的血液大量源。血液成分简单，只有一个循环，颗粒amebocyte，只有三个蛋白质丰富的血浆，血蓝蛋白，C -反应蛋白和α2-巨球蛋白的细胞类型。发自内心的血液收集和离心分离血细胞和血浆。 

视频: Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

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

Using the Horseshoe Crab, Limulus Polyphemus, in Vision Research

The American horseshoe crab, Limulus Polyphemus is one of the oldest creatures on earth, and the animal continues to play an indispensable role in biomedical research. Not only does their blood contain special cells that scientists use to detect bacteriotoxins in our medicines, but their eyes also contain a neural network that has provided much insight about physiological processes operating in our visual system, such as light adaptation and lateral inhibition. The horseshoe crab remains an attractive model for vision research because the animal is large and hardy for an invertebrate, its retinal neurons are big and easily accessible, its visual system is compact and extensively studied, and its visual behavior is well defined. Moreover, the structure and function of the eyes are modulated on a daily basis by a circadian clock in the animal s brain. In short, the visual system of horseshoe crabs is simple enough to be understood yet complex enough to be interesting.
In this video we present three electrophysiological paradigms for investigating the neural basis of vision that can be performed in vivo with Limulus. They are electroretinogram recording, optic nerve recording, and intraretinal recording. Electroretinogram (ERG) recordings measure with a surface electrode the summed electrical response of all cells in the eye to a flash of light. They can be used to monitor the overall sensitivity of the eye for prolong periods of time. Optic nerve recordings measure the spiking activity of single nerve fibers with an extracellular microsuction electrode. They can be used to study visual messages conveyed from the eye to the brain as well as circadian-clock messages fed back from the brain to the eye. Intraretinal recordings measure with an intracellular microelectrode the voltage fluctuations induced by light in individual cells of the eye. They can be used to elucidate cellular mechanisms of retinal processing.

Using the Horseshoe Crab, Limulus Polyphemus, in Vision Research

In this video we perform electroretinogram recording, optic nerve recording, and intraretinal recording with the American horseshoe crab, Limulus Polyphemus. These electrophysiological paradigms can be used for investigating the neural basis of vision in a research or teaching lab.

马蹄蟹，鲎波吕斐摩斯，在视觉研究

The American horseshoe crab, Limulus Polyphemus is one of the oldest creatures on earth, and the animal continues to play an indispensable role in ...

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 ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

在成年斑马鱼的生化分析采血车

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

一种实用的新的方法来提取基因组DNA保存血浆蛋白从血液采集套件

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

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

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

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 ...

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 ...

Sampling Blood from the Lateral Tail Vein of the Rat

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

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

从大鼠的侧尾静脉血液取样

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

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

重复采血车在成年斑马鱼验血

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

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 ...