这项工作是由国家机构的健康1R21HL092370-01（GG），1R01 HL095764-01（GG）; R01HL082694（JW），美国心脏协会的创始人联属公司（0855943D，GG），石溪世界 - 赫伯特基金，纽约（SHP）。

1。准备<ol><li>准备以下的解决方案和管（ 表1）如下： <ol><li> Hanks溶液，无钙，镁或指示器，与青霉素（100单位/毫升）/链霉素（100毫克/毫升）。 </li><li>磷酸盐缓冲盐水（PBS），1个，无钙，无镁。 </li><li>乙醇，70％，500毫升。 </li><li>与PBS缓冲甲醛，7％至10％，使500毫升。 </li><li>麻醉科的解决方案： <ol><li>圣阿韦坦。小心地加入2 - 甲基-2 - 丁醇的5毫升至5克的2,2,2 - 三溴乙醇。一旦溶解后，在室温下在黑暗中保持原液。以0.25毫升原液和稀释1xPBS在玻璃底瓶用10ml。包裹铝箔，地点的瓶在37℃下，直至溶解，然后等分试样分成5毫升的聚丙烯管中，并保存在冰箱中。温暖的一份日上午的实验。 </li><li>巴比妥类药物。与PBS稀释原液2.6％巴比妥类溶胶ution。将聚丙烯管中的3-4毫升的等分试样。 </li></ol></li></ol></li><li>组织三个工作区（ 图1）。 </li><li><ol><li> A区：安排以下项目：的的学习形式记录研究的识别号码，日期，体重，重量右心，左心和隔垫;精密刻度（0-50克精度0.01克），以确定身体的重量;微观尺度以确定的心脏重量精度0.001克的;称量皿;麻醉解决方案（清楚标明）;小杜瓦瓶用液氮;用冰冷的绝热容器;管和24孔板上，以收集血液和组织标本（ 表1）;外科手术器械（ 表2中，图2）;及清洁工具（ 表1）的解决方案。 </li><li> B区：安排以下项目：解剖显微镜，右心导管连接到放大器和笔记本电脑连接到压力控制单元。该导管被放置到含有水的烧杯中。安排手术器械（ 表2），最佳的长度，缝合切件，放入称重船磁带（高压釜磁带是最优的）削减到最佳的长度和宽度，为方便显微镜或实验室工作台一字排开;棉签头发卸妆液。在研究开始时，校准导管。 </li><li> C区：安排以下项目：放大镜镜片;气管插管; 1毫升注射器，缝合切到最佳长度，放入称重船小杜瓦瓶液氮;与冰的绝缘容器;管和24孔培养板，收集BAL和组织标本（ 表1）;外科手术器械（ 表2）;解决方案来执行BAL和，清洁气管插管和工具（ 表1）。 </li></ol></li></ol> 2。右心导管检查<ol><li>用一个精确的刻度称鼠标轻轻将鼠标移动到一个权衡b燕麦。请一定要轻轻地，悄悄地处理动物。记录重量。小鼠颈静脉的直径以容纳压力导管20克或更多，因为所描述的协议最适合静脉导尿较小的小鼠（17克或更多），只要是足够大的，它是可能的。 </li><li>麻醉鼠标通过注射圣阿韦坦取决于体重（10微升/克）， 例如用于20克给予200微升，25克给予250微升，30克给予300微升。的确切的注射体积取决于小鼠品系，需要进行调整。请一定要处理的动物，轻轻地，静静地，因为压力可以改变对麻醉剂的反应。 </li><li>鼠标后镇静剂，不醒人事，双折叠纸巾上。注意：在纸张上的ID号。脱毛乳液轻轻擦到腹侧方面的脖子上，以清除手术的领域。将鼠标在纸张上，等待1-2分钟。 </li><li>删除从腹侧方面​​的发头发对头发的生长方向，用棉签轻轻地抚摸着脖子上。 </li><li>请仔细确定足够的深度麻醉通过检查的脚趾反射的情况下，鼠标没有反应，捏脚趾。 </li><li>快速和轻松的工作环境的权利心脏导管插入术，其余应不超过10分钟，最佳3-6分钟。的组织，这是很重要的，因为不干燥导管需要滑行进入静脉和静脉内的水分的层上移动。一致的定时的过程中是很重要的的产生的数据组间可比性。 </li><li>将鼠标背部的纸巾，用纸巾转移到保丽龙板。修复的爪子和头，用高压釜磁带到位。 </li><li>切开皮肤，从下颚到胸骨（胸骨）。 </li><li>仔细解剖甲状腺隆重以上对下颌骨暴露右颈内静脉一第二气管。 </li><li>广场保丽龙板在解剖显微镜下按住鼠标聚焦平面在地方和镜头的约0.8倍的放大倍率（总放大倍率镜头所述目镜]约8倍。 </li><li>小心地除去周围结缔组织与手术镊子显微切割的右颈静脉。 </li><li>将两个件的缝合线的顶部的右颈静脉。领带最接近的下颌骨的缝合线，以关闭在静脉的血流量在一个狭小的涓流，放置在一个松散的结静脉周围的是最接近胸骨/乳房骨缝合线。一个可以使用的小滴的血在静脉孔后来发现，这应该成为必要。 </li><li>呼吸，放松放松颈部和下巴的肌肉，保持你的眼睛的静脉通过目镜上。这将确保你的手和手指移动安全，顺利和轻松。 ，虽然静脉用钳子在身边最亲近的下颌骨，到两者之间的缝合线用微型剪刀，用另一只手操作的静脉切开一个口子。放下手中的剪刀，并保持该手轻松，觉得自己的导管。轻轻检索拇指和食指（ 图3）之间的导管保持。将导管插入静脉的孔。 </li><li>轻轻推进右心导管插入（ 图3）。观察导管得到多远导管需要被插入的近似指数和观察显示器的标记。一旦压力曲线显示，轻轻地拧紧缝合导管周围。由于导管的抗张强度，给它一个线圈，因为鼠标的呼吸和心脏的跳动增加运动，另外用胶带固定导管。 </li><li>记录压力测量为供以后分析2分钟（ 图4）。 </li><li>打开导管缝合，然后轻轻地retrie已经导管。擦拭的部分的压力换能器的后面，将放入烧杯中，加水。请勿触摸压力传感器。 </li><li>将鼠标顶部纸巾上的区域A的安乐死，收集血液和脾组织。清洁手术器械。 </li><li>启动程序的下一个动物。当定时准许，在等待记录的压力曲线（2.15）注入麻醉。 </li></ol> 3。的补血健脾样品收集<ol><li>注射巴比妥酸盐溶液，400μL每鼠标，等待1-2分钟。这是在我们的实验室进行例行避免，小鼠可能在不经意间恢复从阿佛丁麻醉而拖垮。实验是适当的标准化取得注入相同剂量每只小鼠巴比妥盐溶液，并通过等待相同数量的时间之前，血液收获。如果允许的实验动物护理和使用委员会和只要仔细地监测作为动物麻醉深度的前放血，这个步骤可以省略。 </li><li>切口进腹。打开尾静脉（腔静脉），用移液管收集血液。 </li><li>取出脾脏，脾进入Eppendorf管内，急速冷冻在液氮中的一块，或者进入到一个含有Hanks缓冲液后准备单细胞的24孔板。 </li><li>鼠标转移之上的薄纸收集BAL肺引流淋巴结，肺，心脏组织的工作区域C。清洁手术器械。 </li></ol> 4。肺和心脏标本的收集<ol><li>解剖气管免费的肌肉和结缔组织。配售钳下的气管，拉缝合。轻轻产生足够的拉伸切开一个口子。保持温和的伸展，插入气管插管，用轻微的旋转运动，并缝合套管诠释Ø的地方。插入气管插管，气管确保湿润，如果有必要的Hanks液中加一滴。在整个过程中，呼吸，放松颈部和下巴的肌肉，保持手部动作的安全和顺畅。 </li><li>解剖开的肋骨，取出胸骨开始从腹部末端。 “请勿打扰”的胸部，因为这将使它很难找到淋巴结的内容。 </li><li>轻轻地插入Hanks缓冲液1毫升，进行支气管肺泡灌洗液（BAL），轻轻转动注射器和检索支气管肺泡灌洗液。重复3倍，接近管中，置于冰上。在整个手术过程中，保持眼睛的端部，连接到注射器的气管插管。一目了然的胸部，观察通货膨胀和通货紧缩的肺部。 </li><li>收获肺保持肋条笼形件的壁用一组镊子，寻找和检索与另一镊子的淋巴结的淋巴结。到24孔板的地点淋巴结。这是重要的为k现在要搜寻的位置：从颈部开始，找到从右侧的鼠标的肋条笼形件的入口，并期待脊柱以上（ 图5）。 </li><li>收获的心脏和纸巾。通过仔细解剖旁边的隔膜隔离的心。转移到工作区A称重，记录重量，并放入Eppendorf管在液氮中速冻。 </li><li>将关闭主支气管周围缝合，左肺叶。解剖左肺叶免费，自由的地方到eppendorf管中，急速冷冻于液氮中，或放入24孔板的井购买隔离的单细胞悬浮液中含有Hanks溶液。 </li><li>将约。 0.5毫升缓冲甲醛溶液通过气管插管到剩余的肺叶。通货膨胀后，解剖肺叶的胸部和地方50ml管中含有甲醛溶液。对于一个替代的研究设计，肺部可膨胀与优化正常切削媒体（OCT，用PBS稀释1:4）和在模具中未稀释的OCT冰冻切片后准备冻结。另一项研究设计可能需要获得快速冷冻和肺组织单细胞悬液从肺部。在这种情况下，没有通货膨胀是必要的：解剖其它肺叶;检索它们从成含有的Hanks缓冲液的24孔板的胸部和放入。 </li><li>取出气管插管，用Hanks缓冲液冲洗干净，清洁的手术器械。 </li></ol> 5。右心导管产生的压力曲线分析与没有知识中使用LabChart 7软件的组标识每个记录的记录的右心室压力数据进行分析。超过20条曲线被随机地选择和为每条曲线的最大和最小的心室压力之间的差异的测量（ΔP）。平均ΔP计算给右Ventricular收缩压。

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Res. 52, 432-441 (1983).</li><li>Kugathasan, L., 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=The+angiopietin-1-Tie2+pathway+prevents+rather+than+promotes+pulmonary+arterial+hypertension+in+transgenic+mice.">The angiopietin-1-Tie2 pathway prevents rather than promotes pulmonary arterial hypertension in transgenic mice.</a> J. Exp. Med. 206, 2221-2234 (2009).</li><li>Bearer, C., Emerson, R. K., ORiordan, M. A., Roitman, E., Shackleton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+tobacco+smoke+exposure+and+persistent+pulmonary+hypertension+of+the+newborn.">Maternal tobacco smoke exposure and persistent pulmonary hypertension of the newborn.</a> Environ. Health Persp. , 105-202 (1997).</li><li>Graham, B. B., 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=Schistosomiasis-induced+experimental+pulmonary+hypertension:+role+of+interleukin-13+signaling.">Schistosomiasis-induced experimental pulmonary hypertension: role of interleukin-13 signaling.</a> Am. J. Pathol. 177, 1549-1561 (2010).</li><li>Butrous, G., Ghofrani, H. A., Grimminger, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+vascular+disease+in+the+developing+world.">Pulmonary vascular disease in the developing world.</a> Circulation. 118, 1758-1766 (2008).</li><li>Crosby, 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=Praziquantel+reverses+pulmonary+hypertension+and+vascular+remodeling+in+murine+schistosomiasis.">Praziquantel reverses pulmonary hypertension and vascular remodeling in murine schistosomiasis.</a> Am. J. Respir. Crit. Care Med. 184, 467-473 (2011).</li></ol>

没有利益冲突的声明。

此处列出的实验流程，允许快速和同时测量右心室收缩压和收获的样品分析的反应在肺部，心脏和免疫系统的小鼠。该过程结合心脏生理学测量，微解剖和随后的组织为活细胞研究，组织学分析，或组学分析的组织收获。的完整过程只需不到20分钟，每鼠标。由于工作区域组织的工作流程，2-3的动物可以同时进行研究。因此，程序是适用于小的，有针对性的实验12和设置在很宽的范围内进?...

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td> 2 - 甲基-2 - 丁醇</td><td> Sigma-Aldrich公司</td><td> 152463 </td><td></td></tr><tr><td> 2,2,2 - 三溴乙醇</td><td> Sigma-Aldrich公司</td><td> T48402 </td><td></td></tr><tr><td>消毒肥皂（专题喷雾TB加STERIS） </td><td> Fisher Scientific则</td><td> 1629至1608年</td><td></td></tr><tr><td> 200的证明，乙醇，绝对无水ACS / USP级</td><td> PHARMCO-AAPER </td><td> 111000200 </td><td>用蒸馏水稀释至70％的</td></tr><tr><td>甲醛溶液</td><td> Sigma-Aldrich公司</td><td> F1635-500ML </td><td>稀释至7-10％，预计ldehyde浓度在PBS中浓度为1x使用PBS原液和水</td></tr><tr><td> Hanks溶液，无钙，镁</td><td> Fisher Scientific则</td><td> 21-022-CV </td><td></td></tr><tr><td> OCT </td><td>组织康</td><td> 4583 </td><td></td></tr><tr><td> （10,000 U / ml）的青霉素/链霉素（10000毫克/毫升）解决方案</td><td> Thermo Scientific的</td><td> SV30010 </td><td></td></tr><tr><td>磷酸盐缓冲盐水（PBS），无钙，无镁，1倍和10倍的解决方案</td><td> Fisher Scientific则</td><td></td><td></td></tr><tr><td>戊巴比妥钠26％ </td><td> Fort Dodge动物保健</td><td> NDC 0856-0471-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> 医学实验 </td></tr><tr><td>板12，24，96以及</td><td>鹰</td><td></td><td></td></TR&gt; <tr><td>移液管</td><td> Fisher Scientific则</td><td> 13-711-9BM </td><td></td></tr><tr><td>管，EDTA涂层; </td><td>萨尔斯塔特</td><td> 2013-08 </td><td></td></tr><tr><td>管0.65毫升和1.7毫升的微离心</td><td> VWR </td><td></td><td></td></tr><tr><td>管12×75毫米聚丙烯</td><td> Fisher Scientific则</td><td> 14-956-1D </td><td></td></tr><tr><td>管，各种大小，聚丙烯</td><td> Fisher Scientific则</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> 仪器 </td></tr><tr><td>钳，DUMON第5精细</td><td>精细科学工具</td><td> 11254-20 </td><td></td></tr><tr><td>钳，超等格雷夫-0.5mm的技巧弯曲</td><td>精细科学工具</td><td> 11152-10 </td><td></td></tr><tr><td>钳，额外的网络连接东北格雷夫-0.5mm的提示直</td><td>精细科学工具</td><td> 11150-10 </td><td></td></tr><tr><td> 19 GA 18GA，套管</td><td> BD </td><td>精密的滑翔针</td><td>切到最佳长度，减弱外嘶哑创建一个粗糙的外表面</td></tr><tr><td>剪刀，剪刀剥离超薄刀片9厘米</td><td>精细科学工具</td><td> 14081-09 </td><td></td></tr><tr><td> BAL，编织丝线缝合，缝合4-0 </td><td>精细科学工具</td><td> SP116 </td><td></td></tr><tr><td>缝合右心导管检查，编织丝线缝合，6-0 </td><td>泰利福医疗</td><td> 18020-60 </td><td></td></tr><tr><td>注射器，1毫升</td><td> BD </td><td> 309659 </td><td></td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>放大器，PowerLab系统4/30 </TD&gt; <td> ADInstrument </td><td>型号ML866 </td><td></td></tr><tr><td>导管，压力F1.4 </td><td>米勒仪器公司</td><td> 840-6719 </td><td></td></tr><tr><td>解剖显微镜</td><td>彩色摄影</td><td></td><td></td></tr><tr><td>钳，Vannas春风似剪刀毫米刀片</td><td>精细科学工具</td><td> 15000-00 </td><td></td></tr><tr><td>卤素照明台放大镜</td><td> Fisher Scientific则</td><td> 11-990-56 </td><td></td></tr><tr><td>笔记本电脑</td><td>华硕</td><td>型号A52F酷睿i5处理器，15英寸</td><td></td></tr><tr><td>光源</td><td> Amscope </td><td> HL-250-A </td><td></td></tr><tr><td>压力控制单元</td><td>米勒仪器公司</td><td> PCU-2000 </td><td></td></tr><tr><td>软件，Labchart。亲V.7 </td><td> AD在剂量率仪</td><td></td><td></td></tr></tbody></table>

right ventricular systolic pressure measurements in combination with harvest of lung and immune tissue samples in mice

正确的心脏的功能是把血液通过肺部，从而将右心脏的生理和肺血管生理。炎症的心脏和肺的功能，是一种常见的改性剂，通过制定生产的细胞因子和生长因子，细胞浸润，并开始重塑过程1。 右心室与左心室相比，是一个低压泵工作在一个相对窄的区域的压力变化。增加与肺动脉压力增加的压力在肺血管床和肺动脉高压2。肺动脉高压往往伴有炎症性肺部疾病，如慢性阻塞性肺疾病，自身免疫性疾病3。由于肺动脉高压赋予预后不良的生活质量和寿命，大量的研究是针对了解的机制，mig的HT药物干预4的目标。有效的管理手段，为肺动脉高压的发展面临的主要挑战仍然是复杂的分子和细胞变化在权利的心，肺和免疫系统的同时了解。 在这里，我们提出了一个程序的工作流程，快速，精确的测量压力变化在正确的小鼠心脏的心脏，肺和免疫组织样本的同时收获。该方法是基于直接导管右心室，通过颈内静脉接近上身的小鼠，最早是在20世纪90年代后期，作为替代措施的压力在肺动脉5-13。组织团队的方式有利于快速右心导管检查技术。这使得能够进行测量的小鼠的自发呼吸室内空气。在不同的工作领域的组织的工作流程减少了时间延迟和打开的可能性，同时进行生理实验和收获的免疫系统，心脏和肺组织。 这里列出的程序的工作流程，可以适应各种各样的实验室设置和研究设计，从小型的，有针对性的实验，到大型药物筛选试验。同时采集心肌的生理数据，可以扩展到包括超声心动图5,14-17和收获的心脏，肺和免疫组织减少了需要获得数据的科学知识为基础向前移动的动物。这里介绍的程序的工作流程也提供了理想的基础免疫，肺和心脏功能的网络链接获取知识。这里概述的相同的原则可以适用于研究根据需要的其它的或附加的器官。

获得合适的心脏压力曲线的主要结果是通过合适的心脏导管的正确位置。右心室的导管内部的正确放置的压力时间曲线的形状是重要的，因为会导致压力高原（ 图4）。相反，高低不平的曲线，表明由呼吸或心脏跳动的运动靠在墙上的右心室导管移动。为了检测的阶段的动物的生存的潜在的问题，需要计算ΔP，和心脏的速度的标准偏差。预计这两个值都没有显着不同的治疗组之间。此...

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Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice | Immunology and Infection | Video

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

一个特异，快速的协议，同时探讨合适的心脏功能，肺部炎症和免疫反应，被描述为一种学习工具。视频和数字，描述的生理和显微切割技术在组织团队的方法，是适用于用于小型到大型研究。

右心室收缩压测量组合在小鼠的肺和免疫组织样品的收获

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

right-ventricular-systolic-pressure-measurements-combination-with

Research

Education

JoVE Journal

JoVE Core

Biology

Immunology and Infection

Human Biology

Circulatory and Pulmonary Systems

SCIENTISTS IN ACTION

25.0K 次观看.  New York University School of Medicine, Tuxedo. 一个特异，快速的协议，同时探讨合适的心脏功能，肺部炎症和免疫反应，被描述为一种学习工具。视频和数字，描述的生理和显微切割技术在组织团队的方法，是适用于用于小型到大型研究。

视频: Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

科研

免疫与感染

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic Poly(I:C)

Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric disorders. Numerous studies have demonstrated that MIA model is able to show face, construct, and predictive validity that are relevant to autism and schizophrenia. To model MIA, investigators often use viral mimic polyinosinic:polycytidylic acid (poly(I:C)) to activate the immune system in pregnant rodents. Generally, the offspring from immune activated dam exhibit behavioral abnormalities and physiological alterations that are associated with autism and schizophrenia. However, poly(I:C) injection with different dosages and at different time points could lead to different outcomes by perturbing brain development at different stages. Here we provide a detailed method of inducing MIA by intraperitoneal (i.p.) injection of 20 mg/kg poly(I:C) at mid-gestational embryonic 12.5 days (E12.5). This method has been shown to induce acute inflammatory response in the maternal-placental-fetal axis, which ultimately results in the brain perturbations and behavioral phenotypes that are associated with autism and schizophrenia.

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic PolyI:C

Maternal immune activation (MIA) is a model for an environmental risk factor of autism and schizophrenia. The goal of this article is to provide a step-by-step procedure of how to induce MIA in the pregnant mice in order to enhance the reproducibility of this model.

母体免疫激活诱导小鼠的病毒性模仿聚妊娠中期阶段（I：C）

Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

人精切肺切片中物质的细胞毒性及免疫调节作用的评价

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

在小鼠小腿上应用一致的按摩样扰动，并监测由此产生的肌肉压力变化

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

由加德纳菌阴道膀胱暴露在小鼠中引发的复发性大肠杆菌尿路感染

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples

Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical challenge. Among the ~600 proteases encoded by the human genome, neutrophil serine proteases (NSPs) are thoroughly investigated for their involvement in the onset and progression of inflammatory conditions including respiratory diseases. Uniquely, secreted NSPs not only diffuse within extracellular fluids but also localize to plasma membranes. During neutrophil extracellular trap (NETs) formation, NSPs become an integral part of the secreted chromatin. Such complex behavior renders the understanding of NSPs pathophysiology a challenging task. Here, detailed protocols are shown to visualize, quantify and discriminate free and membrane-bound neutrophil elastase (NE) and cathepsin G (CG) activities in sputum samples. NE and CG are NSPs whose activities have pleiotropic roles in the pathogenesis of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD): they promote tissue remodeling, regulate downstream immune responses and correlate with lung disease severity. The protocols show how to separate fluid and cellular fraction, as well as the isolation of neutrophils from human sputum for enzymatic activity quantification via small-molecule F&#246;rster resonance energy transfer-based (FRET) reporters. To gather specific insights into the relative role of NE and CG activities, a FRET readout can be measured by different technologies: i) in vitro plate reader measurements allow for high-throughput and bulk detection of protease activity; ii) confocal microscopy spatiotemporally resolves membrane-bound activity at the cell surface; iii) small-molecule FRET flow cytometry enables for the rapid evaluation of anti-inflammatory treatments via single-cell protease activity quantification and phenotyping. The implementation of such methods opens the doors to explore NSPs pathobiology and their potential as biomarkers of disease severity for CF and COPD. Given their standardization potential, their robust readout and simplicity of transfer, the described techniques are immediately shareable for implementation across research and diagnostic laboratories.

The protocols herein described provide a guide to visualize and quantify the activity of neutrophil proteases in human sputum. The applications of such analysis span from the evaluation of anti-inflammatory treatments, to biomarker validation, drug screening and large cohort clinical studies.

监测人类痰样中的中性粒细胞和导管素 G 活性

Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

传染性单核细胞增多症儿童外周血样本中免疫细胞亚群的分离

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model

Periodontitis (PD) is a highly prevalent, chronic immune-inflammatory disease of the periodontium, that results in a loss of gingival soft tissue, periodontal ligament, cementum, and alveolar bone. In this study, a simple method of PD induction in rats is described. We provide detailed instructions for placement of the ligature model around the first maxillary molars (M1) and a combination of injections of lipopolysaccharide (LPS), derived from Porphyromonas gingivalis at the mesio-palatal side of the M1. The induction of periodontitis was maintained for 14 days, promoting the accumulation of bacteria biofilm and inflammation. To validate the animal model, IL-1&#946;, a key inflammatory mediator, was determined by an immunoassay in the gingival crevicular fluid (GCF), and alveolar bone loss was calculated using cone beam computed tomography (CBCT). This technique was effective in promoting gingiva recession, alveolar bone loss, and an increase in IL-1&#946; levels in the GCF at the end of the experimental procedure after 14 days. This method was effective in inducing PD, thus being able to be used in studies on disease progression mechanisms and future possible treatments.

Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model

In this study, a rat model of induction of periodontitis is presented via a combination of retentive ligature and repetitive injections of lipopolysaccharide derived from Porphyromonas gingivalis, over 14 days around the first maxillary molars. The ligation and LPS injection techniques were effective in inducing peridontitis, resulting in alveolar bone loss and inflammation.