我们要感谢保罗威格尔在大学的博士。俄克拉何马州30单克隆抗体检测HARE/Stabilin-2受体和她的技术援助，珍妮特威格尔。这项研究是由内布拉斯加大学研究基金资助。

1。肝切除术（PO Seglen 12和R Blomhoff 13 14,15与修改通过） <ol><li>添加10毫升30％的异氟醚聚乙二醇在一个封闭的5.5大号室培养皿中。等待10-15分钟后，异氟醚除了内室创造一个稳定的气氛，这是最佳。 </li><li>老鼠放置在密封腔，并使其成为麻醉。麻醉的全面影响是显而易见的，当动物成为跛行，反应迟钝，和展品深呼吸。 </li><li>放置2毫升异氟醚聚乙二醇在30 mL注射器管底部的一些棉球。 </li><li>放在尿布覆盖的托盘，并将其放置在口鼻注射器管的大鼠在它的后面。 </li><li>立即证实，用70％乙醇润湿腹部深麻醉。不要让动物死亡异氟醚过量。 </li><li>使用绷带剪刀和镊子，揭露整个ABDominal腔。 </li><li>找到腔PORTA，使用产钳，吸引两股的手术丝线或涤纶线（结扎）下腔以上肠系膜分支立即PORTA。 </li><li>撤回镊子和领带一松上手结。 </li><li>使用下面的腔PORTA钳轻轻拉了回来，以理顺静脉，cannulate Insyte Autoguard导管（1.3 GA，18 × 300毫米，BD​​公司）或类似的导管静脉。导管应超越由线程组成的环几个毫米。 </li><li>缩回，删除通过导管释放弹簧加载触发针。血液应该开始渗入指示正确的结扎安置导管。 </li><li>拧紧上手结和安全与另一上手结。 </li><li>要么切断下腔静脉或降主动脉（主动脉abdominalis），让血液循环系统排出。 </li><li>开始冲洗肝脏含氧TBS电视台在37 ° C，移除在20毫升/分钟的流速的血液（漂白）。肝脏变成一个壤土颜色从紫红色。 </li><li>而肝脏是潮红，切除肝脏切掉消化道和结缔组织。重要的是不要划破Glisson囊肝或穿刺。 </li><li>放置在超过一个漏斗，允许缓冲区来收集和再循环的塑料网肝脏。在这一点上的缓冲区不应再循环，但流入废物烧杯。法拉盛不应持续超过10分钟。 （ 图2A）。准备步骤2.1所需的内吞作用的解决方案。 </li></ol> 2。国际化125 - SA - B - HEP（或其他适当的标记配体） <ol><li>至50 ml的RPMI媒体在小烧杯中，加入终浓度为0.05％BSA和0.01 mCi的125 I - SA - B - HEP或其他适当标记的配体的内吞作用。 </li><li>将烧杯的apparatus到允许充氧。 </li><li>关闭泵，开关入口管，然后打开泵在20毫升/分钟。 </li><li>作为标记的RPMI方法肝脏，关闭泵，并让多余的液体在漏斗和油管漏。 </li><li>媒体烧杯放入漏管允许再循环标记的媒体，然后重新启动泵。 </li><li>让液体通过肝脏不超过1小时循环。 </li><li>在此期间内化步骤，确保在肝脏的温度是通过覆盖它，并准备胶原酶消化稳定。 </li></ol> 3。胶原酶消化肝脏消化<ol><li>新鲜溶解并过滤（0.45微米）胶原酶（100毫克/公斤体重大鼠）应添加到缓冲区的总体积不超过60毫升，在37 ° C。量是依赖于管的长度/内部容积，应作相应调整。 </li><li>停泵和交流从媒体烧杯的TBS入口管。 </li><li>冲洗2分钟，在50 mL / min的钙依赖的桥粒细胞路口松动允许肝脏。 </li><li>而肝脏是潮红，交流媒体烧杯中含有胶原酶烧杯仪器。 </li><li>冲洗后，立即开始在流速为20 mL / min的长达15分钟的消化胶原酶在循环回路。由于胶原酶批次不同批号，在消化的数量和时间的变化，需要为每一个新的胶原酶很多优化。胶原酶也是钙依赖。关闭泵和切换缓冲区和天然气线从保温瓶一个合剂B.转移废物容器的污水合剂乙线（ 图2B）。 </li><li>停泵，取下导管和肝脏转移到一盘菜含有20-30毫升缓冲1。 </li><li>剥开Glisson囊肝和动摇的细胞出去了在液体中。 </li><li>由于液体变为不透明的细胞物质，通过100微米过滤通过一个30微米网格，然后到50 mL锥形冰网格传输的细胞。 </li><li>添加更多的缓冲区1到肝脏，并继续摇晃肝基质细胞，网状过滤器和锥形转移到液体。 </li><li>重复步骤3.7至3.9，直到没有更多的细胞可以脱落合理的肝晃动。 </li></ol> 4。肝细胞和SEC的净化<ol><li>离心机50毫升含3分钟，在150 XG细胞conicals。颗粒的肝细胞。 </li><li>转移包含非实质细胞，新鲜的50毫升冰conicals的液体部分。 </li><li>洗肝resuspending在缓冲区3沉淀，并重复步骤4.1和4.2三次。 </li><li>洗末，颗粒≥97％纯肝细胞。为了获得SECS，离心管C的所有ontaining池上清液在10分钟的200 XG（含造血干细胞，秒，以及KCS和一些小肝）。在4 ° C </li><li>吸缓冲区和4颗粒重新悬浮于5 mL的RPMI / BSA ° C。 </li><li>池的细胞聚集成2管，并添加35毫升的体积的RPMI / BSA的最多。 </li><li>离心3分钟，在100 XG conicals。 </li><li>收集前25毫升的液体转移到一个干净的50 mL锥形。 </li><li>重悬沉淀在余下的10毫升媒体，并添加25毫升冷的RPMI / BSA和离心3分钟，在100 XG。 </li><li>再次重复步骤3.8和3.9，弃细胞沉淀和低10毫升媒体。 </li><li>离心supernatents颗粒秒，KCS，造血干细胞在10分钟200 XG。在4 ° C </li><li>准备三个50毫升20毫升25％Percoll上冰的PBS管。底图与15毫升50％，每管Percoll。 </li><li>在总体积为30 mL的RPMI / BSA重悬细胞沉淀。 </li><li>仔细叠加选管会10毫升的细胞和离心20分钟，在900 XG h梯度在4 ° C </li><li> SECS和KCS有着非常密切的浮力密度和25/50％界面。造血干细胞通常在25％/媒体接口由于其较高的脂质储存细胞的浮力。任何剩余的肝细胞和血细胞降低buoyancies颗粒。吸从25/50％界面附近的自上而下和丢弃这种材料。 </li><li> 25/50％接口转移到与冷的RPMI新鲜锥形收集细胞。最终体积应〜40毫升，以稀释的Percoll。 </li><li>锥形离心10分钟350 XG在4 ° C沉淀细胞。 </li><li> 〜10毫升预热R​​PMI酸洗玻璃培养皿中含有100 U /毫升Penicillin/100克/ ml链霉素和地方细胞或结晶盘重悬细胞。 </li><li>孵育15分钟的细胞。在37 ° C，在一个组织文化的孵化器。 </li><li>岩石或旋流板一点点然后收集上清SECS。 KCS坚持比秒更迅速地在玻璃上。此外，SECS可从KCS分隔，使用抗CD31或anti-Stabilin2/HARE抗体结合磁珠immunopurification。 </li><li>台盼蓝排除使用hemacytometer或使用自动细胞计数器的生存能力和细胞数量可能被评定。 </li><li>文化的SECS纤维连接蛋白涂层的塑料菜在37 ° C，5％CO 2，与血管内皮生长因子为40毫微克/毫升，100 U / ml青霉素，100μg/ mL链霉素或牛血清白蛋白与肝细胞条件培养基RPMI/0.25 ％。 </li><li>肝细胞，KCS和SECS标记材料的内化的可评估使用伽玛计数器和规范使用这些培养方法细胞总蛋白或显微镜（荧光标记）。 </li></ol> 5。代表性的成果: 肝细胞净化≥97％和证券交易委员会的净化是表型盟友≥95％，使用这种方法。腹腔，门静脉插管，热烫的肝切除，都应该出现在一分钟内获得最佳效果。 HARE/Stabilin-2是对肝脏SECS的特异性受体，并不表示在其他类型的肝细胞。在此示例中，肝和SECS细胞裂解物5％的SDS - PAGE分离，并用单克隆抗体检测对Stabilin - 2两种异构体（ 图3） 。 普通肝素注入血液流是由肝脏清除16。间隙是主要由肝脏SECS肝细胞和KCS 17 。 Stabilin-2/HARE受体是主要清除受体结合和内化秒18的肝素。在我们的例子中，我们标记羧酸葡萄糖/ NS组与生物素标记的肝素。碘化链亲和素生物素是共轭化的肝素PROT检测eoglycan。 ，随后由放血标记的肝素注射，注射后30分钟，让我们监察机关内在这种形式的肝素。在这短短的时间间隔，肝的主要通关器官（ 图4）。 为了更清楚地了解细胞类型是负责清除，我们增加了125 I - SA - B - HEP辅以0.05％BSA的RPMI媒体，并允许它通过肝脏循环20分钟。胶原酶消化和蜂窝净化，绝大多数标记肝素内肝细胞（ 图5）SECS。 <img src="/files/ftp_upload/3138/3138fig1.jpg" alt="图1"/> 图1基本的肝架构。 SECS的血窦形成的墙壁通常是孔（小群各界）。星状细胞（HSC）被发现在Disse间隙库普弗彗星英语学习者（KCS），它们通常存在于血窦微血管。肝Disse间隙中占有的另​​一边。 <img src="/files/ftp_upload/3138/3138fig2.jpg" alt="图2"/> 图2。灌注装置的示意图。一）设置设备在冲洗/洗肝血窦。 TBS是在1升容量瓶中。二）一个125〜60 ml缓冲液2 mL容量瓶中含有胶原酶用于消化肝脏，在一个封闭的电路。污水线也发生了变化，从废弃物容器，烧瓶乙通过削减橡胶瓶塞的缺口。氧行不浸入含胶原酶（乙烧瓶）的缓冲区，以避免起泡。在每个烧瓶塞子​​的缺口，让污水线的有效转移，以防止氧气的压力增加。 <img src="/files/ftp_upload/3138/3138fig3.jpg" alt="图3"/> 图3。 </s仲&gt;肝细胞的准备工作是不污染秒。从肝细胞的纯化批次（泳道1）和秒（巷2）细胞裂解液等量的5％的SDS - PAGE分离，涂抹到硝酸纤维素。单克隆抗体＃30这是对HARE/Stabilin-2两种异构体（315 kDa和190 kDa的）具体是用来探测的裂解物。 <img src="/files/ftp_upload/3138/3138fig4.jpg" alt="图4"/> 图4。标记在机关肝素分布。大鼠经尾静脉注射125 I - SA - B - HEP等待30分钟，然后exsanguinated。采血，以便不给任何机关虚高读数。 （CPM）的每一个器官，每分钟的计数正常化对器官的重量以克。 <img src="/files/ftp_upload/3138/3138fig5.jpg" alt="图5"/> 图5:在肝和SECS标记的肝素量。转速我媒体载有125余SA - B - HEP被允许通过一个完整的肝胶原酶消化和蜂窝净化20分钟分发。等量的肝细胞和SEC细胞蛋白裂解物进行了量化与Bradford法和伽玛计数器计数。 <img src="/files/ftp_upload/3138/3138fig6.jpg" alt="图6"/> 图6。Kupffer细胞分离SECS粘附在玻璃上。细胞被放置在一个酸洗玻璃结晶菜，并坚持15分钟在37℃，5％的CO 2 。含有非坚持细胞的上清轻轻众说纷纭，放在离心管。从玻璃上都坚持细胞（泳道1）非坚持细胞（泳道2）8％的SDS - PAGE分离裂解液，涂抹，并与抗体对CD163 Kupffer细胞的具体探讨。 <img src="/files/ftp_upload/3138/3138fig7.jpg" alt="图7"/> 纤维连接蛋白涂层的塑料上镀图 7秒6的菜肴孵育过夜，0.1％BSA的RPMI补充。 （一）100X（二）200X放大倍率相位对比图像。

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Res. 99, 444-449 (1976).</li><li>Graupera, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinusoidal+endothelial+COX-1-derived+prostanoids+modulate+the+hepatic+vascular+tone+of+cirrhotic+rat+livers.">Sinusoidal endothelial COX-1-derived prostanoids modulate the hepatic vascular tone of cirrhotic rat livers.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 288, 763-770 (2005).</li><li>Yannariello-Brown, J., Zhou, B., Ritchie, D., Oka, J. A., Weigel, P. H. <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+ligand+blot+assay+detects+different+hyaluronan-binding+proteins+in+rat+liver+hepatocytes+and+sinusoidal+endothelial+cells.">A novel ligand blot assay detects different hyaluronan-binding proteins in rat liver hepatocytes and sinusoidal endothelial cells.</a> Biochem. Biophys. Res. Commun. 218, 314-319 (1996).</li><li>Duryee, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide+is+a+cofactor+for+malondialdehyde-acetaldehyde+adduct-mediated+cytokine/chemokine+release+by+rat+sinusoidal+liver+endothelial+and+Kupffer+cells.">Lipopolysaccharide is a cofactor for malondialdehyde-acetaldehyde adduct-mediated cytokine/chemokine release by rat sinusoidal liver endothelial and Kupffer cells.</a> Alcohol Clin. Exp. Res. 28, 1931-1938 (2004).</li></ol>

没有利益冲突的声明。

麻醉大鼠悬滴法在异氟醚蒸气诱导昏迷，是我们研究的首选方法。一个蒸发器，也可能被用来代替注射器与棉花维持在会议厅外的动物，但手术过程中的那么快，它不是必需的。聚乙二醇添加到异氟醚，以减少蒸汽压力和蒸发速度。异氟醚蒸气太多太快会诱发死亡。如果动物死亡前肝脏是空心的，用TBS，血液可能汇集在肝脏血块烫一下，防止血窦和胶原酶消化高效清洗。肝素除可作为抗凝剂，但不是在这些研究中使用，因为我们用我们的探头标记的肝素。 Gey的缓冲盐溶液（GBSS）往往是取代其他实验室秒净化。虽然它是非常有用的SEC的净化，HEPatocytes不容忍GBSS以及缓冲器1-3和存活率都差不多高。当测量的内吞作用，这是很好的做法，措施，在机关和纯化的肝细胞的背景水平。 这种方法的二分之一以下胶原酶肝脏灌注SECS高效净化。另一种方法是由淘洗离心19 Percoll梯度分离非实质细胞。使用超过Percoll梯度淘洗的优点是稍高的细胞存活率和数字。的缺点是，淘洗离心需要一个专门的转子，专用离心机，蠕动泵成本几万块钱。这种方法只需要使用冷藏表离心机，蠕动泵，这是许多实验室的标准。 SECS和选择性粘附KCS的分离是一种标准的方法20日至22日。秒，将坚持以玻璃和塑料虽然这是事实，关键的一点是使用不超过15分钟的孵化酸冲洗干净玻璃，在37 ° C时，5％的CO 2。 KCS将始终坚持比秒的速度更快，最好是轻轻用媒体的KCS提取任何剩余的，已成为松散连接SECS。链霉蛋白酶和其他蛋白酶也省略了在本议定书中的胶原酶消化步骤，增加细胞的存活率。蛋白酶消化细胞外受体，并可能抑制细胞粘附在最后的纯化步骤。 这里介绍的方法具有广泛的应用。虽然我们显示肝素初步过关，获得原代肝细胞肝脏灌注的最终结果，KC，秒，造血干细胞可能是有用的研究，涉及的代谢途径，免疫调节，清道夫活动，及其他数生理studiES。在此过程中最困难的部分是良好的插管，消化，肝脏和肝脏的处理，而无需来自门静脉的导管滑。

<ul><li>缓冲区1:氯化钠6.7毫米氯化钾，142毫米10毫米的HEPES，1.5％BSA，pH值7.4 </li><li>缓冲区2（灌注缓冲区）:67.0毫米氯化钠，6.7 mM的氯化钾，4.8毫米氯化钙2 - H2O，101.0 mM的HEPES，BSA 1.5％，pH值7.4。 </li><li>缓冲区3:137毫米氯化钠，氯化钾4.7毫米，0.7毫米硫酸镁4，1.2毫米氯化钙2 · 2H 2 O，10毫米的HEPES，1.5％BSA，pH值7.4 </li><li> RPMI / BSA:15克/ L，BSA的RPMI </li><li> TRIS缓冲液（TBS）:154 mM氯化钠，10毫米Trizma基盐。 </li><li>胶原酶:胶原酶一个罗氏（＃11088785103），沃辛顿生化或胶原酶我的胶原酶I型（＃LS004691）（＃C0130）或IA（＃C9891）自Sigma - Aldrich。 </li></ul>所有盐自Sigma - Aldrich <table border="1"><tbody><tr><td>名称</td><td>公司</td><td>型号/目录＃ </td><td>评论</td></tr><tr><td> 20L水浴</td><td> ThermoFisher </td><td> 2231 </td><td>水是约45 ° C，以弥补冷却</td></tr><tr><td></td><td></td><td></td><td>管内。 </td></tr><tr><td>蠕动泵</td><td>科尔 - 帕默</td><td> 7553-70 </td><td>的Masterflex系列</td></tr><tr><td>冷冻离心机</td><td> Sorvall </td><td>联想XTR </td><td></td></tr><tr><td>导管</td><td> BD公司</td><td> 381444 </td><td></td></tr><tr><td> Dessicator室</td><td>费舍尔</td><td> 08595E </td><td>使用内部板</td></tr><tr><td> Percoll </td><td>西格玛</td><td> P4937 </td><td></td></tr><tr><td>牛血清白蛋白</td><td> SeraCare </td><td> AP - 4510 - 01 </td><td></td></tr><tr><td> 100微米网状</td><td>光谱实验室</td><td> 146488 </td><td></td></tr><tr><td> 30微米网状</td><td>光谱实验室</td><td> 146506 </td><td></td></tr><tr><td> RPMI 1640培养</td><td> Invitrogen公司</td><td> 21870 </td><td></td></tr><tr><td>聚乙二醇</td><td>光谱实验室小号</td><td> PO107 </td><td></td></tr></tbody></table>

in vivo liver endocytosis followed by purification of liver cells by liver perfusion

肝脏是哺乳动物体内的代谢中心，并作为血液过滤器。肝脏的基本架构说明图1中，其中超过85％的肝脏肿块是由肝细胞和其余15％的细胞质量是枯否细胞（KCS），星状细胞（HSC）组成，正弦内皮细胞（秒）。 SECS形成肝脏内的血管壁，称为窗孔在细胞质内含有专门的形态。细胞质开窗孔的外观（〜100微米）的细胞内秒的行为，使筛子其中最乳糜微粒，乳糜微粒残余物和大分子，但不是细胞，通过向肝细胞和造血干细胞 1（图1）。由于缺乏基底膜，SECS和肝细胞之间的差距Disse间隙。造血干细胞占据这个空间，并发挥了突出的作用，在调控和应对我njury，贮存维甲酸和免疫调节的肝2。 SECS是身体endocytically最积极的显示阵列上其细胞表面的 3的清道夫受体细胞。这些措施包括Stabilin SR - A - 1和Stabilin - 2。一般来说，小的胶体颗粒小于230纳米的大分子缓冲阶段秒，而，大颗粒和吞噬细胞碎片是由KCS 4（吞噬） 。因此，如从血液中糖胺外物质的散装间隙很大程度上取决于对健康和 5,6秒内吞功能。例如，增加血液中的透明质酸水平是指示性的肝脏疾病，从轻微到更严重的形式 7 。 随着一个报告8外，还有SEC存在没有永生细胞株。这永生细胞株，即使是去分化ated，它不表达清道夫受体（我们的数据，未显示）的主要SECS。所有细胞生物学的研究，必须从动物新鲜获得的原代细胞。不幸的是，SECS去分化标准培养条件下，必须在1或2天从动物隔离后使用。 Stabilin - 2的表达或野兔受体 9，CD31，存在细胞质开窗 1秒分化标记。除了 ​​血管内皮生长因子的培养基，或在肝细胞条件培养基培养10,11细胞，可以延长秒分化。 在这份报告中，我们将展示完整的器官内吞活动的SECS使用无线电标记的肝素，透明质酸为美国证券交易委员会的具体Stabilin - 2受体。然后，我们将净化灌注肝脏的肝细胞和SECS来衡量的内吞作用。

Watch this Scientific Journal Video about In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion at JoVE.com

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

从动物获得的无细胞系存在的主要细胞，肝窦内皮细胞（秒）的研究必须执行。这种方法依赖于肝的消化和差速离心SEC随后的培养和实验净化。

在体内肝细胞的分离纯化的肝细胞内吞其次是肝脏灌注

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

in-vivo-liver-endocytosis-followed-purification-liver-cells-liver

Research

JoVE Journal

Biology

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

18.9K 次观看.  University of Nebraska, Lincoln. 从动物获得的无细胞系存在的主要细胞，肝窦内皮细胞（秒）的研究必须执行。这种方法依赖于肝的消化和差速离心SEC随后的培养和实验净化。

视频: In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

分离的CD133 +肝干细胞的克隆扩增

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

科研

生物学

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

大鼠门户成纤维细胞的分离<em&gt;原位</em&gt;肝脏灌注

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

人肝细胞通过一个两步胶原酶灌注过程隔离

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

协议的慢病毒转导与肠组织体下游分析

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

一个标准化的方法肝窦内皮细胞的分析及其窗孔用扫描电子显微镜

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析:在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

小鼠肝脏中细胞类型特异性基因表达分析

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...