NS64571由赠款支持这项工作是从NINDS（CLH），由早期的职业生涯从梅奥诊所（CLH）发展奖，由唐纳德和弗朗西斯Herdrich（CLH）的厚礼。我们想感谢帮助梅奥诊所的流式细胞仪核心。

1。颅内病毒注射液: 下面的技术已被修改，并广泛利用我们的实验室和他的同事。简单地说，丹尼尔的应变Theiler鼠脑脊髓炎病毒（TMEV）或假感染颅内注射（1，10）是年轻小鼠（最好5-6周龄）引起脑浸润白细胞（BILs）。请注意，结果会有所不同，丹尼尔的应变，豆株，GDVII应变之间。对于采伐BILs的目的，小鼠收到2X10 TMEV 5 PFU和感染24小时。 <ol><li>将注射针头（27压力表，¼，肯德尔），1毫升汉密尔顿注射器自动设置提供10μL的病毒。 </li><li>绘制在10μL的DMEM培养液进入注射器小心注意气泡TMEV。在适当的时候，深水感染小鼠获得无病毒的DMEM 10μL。 </li><li>就在注射之前，倒入一个钟罩，包含棉花基板下方的线网格约1毫升的异氟醚。 </li><li> direclty放置在钟罩上线电网的老鼠，直到他们变得轻轻麻醉，脚趾捏不敏感和吸入麻醉固定，约10-15秒。老鼠不应与异氟醚的直接接触，作为物质可刺激性和腐蚀性。 </li><li>虽然在麻醉状态下，准备通过快速定位在适当的坐标上使用剃须和墨的头骨额叶皮层区域的小鼠注射。注射的位置一般是1毫米，前囟和1毫米，横向到上矢状缝右侧（图1）。虽然立体定向注射可适当对一些实验的情况下，我们发现，徒手注射液是没有剃须或墨水适用于大多数实验。临别沿两个轴的进针点皮毛适当的注射。 </li><li>为了使注射，东方针垂直于骨的头骨，并通过按约3毫米的深度。 </li><li>快递按汉密尔顿注射器按钮并等待2秒钟，病毒进入大脑前撤回病毒进入大脑。 </li><li>小心取出针和鼠标放置在干净，干燥的笼。老鼠唤醒麻醉迅速，并在几分钟之内恢复正常功能。适当注意小鼠表现出异常的症状，如注射部位出血过多的动物实施安乐死，可能是适当的。必须遵循正确的动物处理和遵守的美国国立卫生机构动物护理和使用委员会的准则研究院。 </li></ol> 2。脑浸润白细胞细胞制备: <ol><li>准备橡树岭圆底离心管中，其中可容纳10毫升的RPMI 1640毫升Percoll，9和1 ml 10X PBS和管应该坐在板凳上，在室温下在大脑切除过程中30毫升的容量。 </li><li>室温（RT），聚蔗糖帕确加。 </li><li>通过修改异氟醚过量5安乐死的动物，迅速 ​​取出大脑使用不锈钢组织锅铲和地方在15毫升锥形管含冰5毫升的RPMI 。在某些情况下，PBS灌注可适当删除循环BILs准备从外周血细胞。应充分和适当的麻醉前以PBS灌注小鼠。 </li><li>转移大脑和RPMI解决7毫升玻璃耐热品牌Tenbroeck组织磨床和10-15杆轻轻同质化。 </li><li>吸取到匀浆和吸管和向下两次额外的5毫升的RPMI。转让脑匀浆（约10毫升）先前准备的圆底管，轻轻颠倒2-3次混合。 </li><li>在F0360固定角度转子在贝克曼Allegra的X - 22R的临床离心机在室温为30分钟， 旋转 7800克大道的脑组织匀浆。 </li><li>离心后，去除浮动梯度顶部的髓鞘碎片。收集白细胞层以上的红血细胞沉淀（图2）浮。 </li><li>通过一个40微米的细胞过滤器的应变白细胞层到50毫升锥形。解决方案，以50毫升与RMPI量。 </li><li>旋转管含有稀释的细胞悬液，在室温下5分钟在1500转（600克 AVE）的临床离心机。 </li><li>吸弃上清液，收集细胞沉淀。悬浮颗粒在1毫升流式细胞仪缓冲区（1％牛血清白蛋白，0.02％，钙的叠氮化钠和镁无PBS）和转移的细胞悬液5 ml圆底流式细胞仪管。 </li><li>小心地用1 ml聚蔗糖帕确加底图停牌。 </li><li>转速2500 RPM（1400了）在临床离心25分钟，在室温下没有刹车管。 </li><li>白色，蓬松层，用1毫升吸管和转移到新的5毫升流式细胞仪管的界面（图2）删除。洗净彗星英语学习者与4毫升流式细胞仪缓冲。 </li><li>临床离心沉淀细胞在室温5分钟旋转1500转（600克AVE）管。 </li><li>吸上清，收集细胞沉淀。重悬在适当的缓冲液中白细胞颗粒，并保持在新的流式细胞仪管冰。 </li><li>计数细胞，通过台盼蓝拒评估细胞活力，流式细胞仪分析。在一般情况下，调查人员可以期望获得大约5 × 10 5细胞/动物。 </li></ol> 3。送流式细胞免疫分型<ol><li>经过分离和计数白细胞，自旋为3分钟在1500转（600 了 ），在临床离心机的细胞。 </li><li>重悬沉淀阻止缓冲区，其中包含以下:流式细胞仪的缓冲区，2.4G2杂交瘤细胞（FC块; anti-CD16/32）上清和胎儿比例的10时05分01秒的牛血清，孵育30分钟，在4 ° C 。 </li><li>准备和30分钟，在浓度为1:200，并培育，阻止细胞在4 ° C黑暗环境中添加共轭对细胞外抗原的抗体。 200μL的细胞染色法和细胞培养在96孔V型底板。适当的控制，包括未染色细胞和荧光补偿样本，作为必要的。 </li><li>转速1500 RPM在临床离心机转子适当使用96孔板（600 了 ），在室温下3分钟的染色细胞。 </li><li>吸上清，洗与200μL的流式细胞仪缓冲细胞轻轻吹打向上和向下的3倍。 </li><li>重复两次以上的洗涤技术。 </li><li>洗涤后，重悬在2％多聚甲醛和流式细胞仪管转移到细胞。固定是需要流生物安全等级2试剂感染细胞的流式细胞仪分析。 </li><li>以下修改后的流式细胞仪检测方法8运行在BD流式细胞仪刀魂固定细胞和分析脱机使用FlowJo，WinMDI或其他可用的流式细胞仪分析软件的文件。 </li><li> 50-100,000每个样品的事件分析，通常需要足够的免疫表型。 </li></ol>在这个实验中直接使用共轭抗体: CD45的发现与克隆30 - F11。 Ly6C / G的检测与克隆GR1，RB6 - 8C5。 CD11b的发现与克隆M1/70。 F4/80的发现与克隆BM8。 4。代表性的成果: 我们细胞分型结果表明，在感染后24小时（图3）鼠标BILs。 PerCP标记的抗CD45的小鼠白细胞染色检测免疫细胞，PE标记的抗鼠Ly6C / G来检测检测细胞的炎症单核细胞，APC标记的抗鼠CD11b和APC标记的抗鼠F4/80单核细胞谱系。一个BD流式细胞刀魂仪进行分析。 <img src="/files/ftp_upload/2747/2747fig1.jpg" alt="图1"/> 图1。注射部位的解剖定位，注射部位位于前囟和1毫米，1毫米，横向上矢状缝右侧。 <img src="/files/ftp_upload/2747/2747fig2.jpg" alt="图2"/> 图2。最初是直接收集髓鞘碎片层以下和以上的红细胞在Percoll梯度颗粒梯度分离的白细胞和BILs插图。白细胞。白色，蓬松层接口（BILs）收集从聚蔗糖梯度。 <img src="/files/ftp_upload/2747/2747fig3.jpg" alt="图3"/> 图3。从大脑颅内感染后24小时，准备从动物组织匀浆差密度离心分离出脑infilatrating白细胞在24小时后感染小鼠白细胞的免疫表。与荧光标记的抗体对小鼠CD45的细胞染色，Ly6C / G，CD11b的，和F4/80。初始浇注策划CD45，免疫细胞的标志物，对前向散射（FSC），细胞大小的指标（三）执行。 CD45 您好 （A，D），CD45 LO（B，E），和CD45的NEG（F，G）人口的单核细胞和巨噬细胞标记Ly6C F4/80 / G（A，B的相对表达水平分析，F）和CD11b（D，E，G）。 Ly6C / G + F4/80 +细胞CD11b +，CD45您好人口（A，D），从外围这些细胞浸润炎症单核细胞中发现的几乎完全。相比之下，Ly6C/G-F4/80 LO细胞CD11b +巨噬细胞几乎全部被发现在CD45的LO（B，E）和CD45 阴性 （G）的人群，与居民的表型一致。在无数次的实验，我们从来没有观察到大脑未受感染或假感染您好CD45的细胞或Ly6C / G + F4/80 +细胞CD11b +炎症单核细胞特德小鼠（数据未显示）。

<ol>
	<li>Buenz, E. J., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picornaviruses+and+cell+death.">Picornaviruses and cell death.</a> Trends Microbiol. 14, 28-36 (2006).</li><li>Buenz, E. J., Limberg, P. J., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+3-parameter+flow+cytometry-based+cell+death+assay.">A high-throughput 3-parameter flow cytometry-based cell death assay.</a> Cytometry A. 71, 170-173 (2007).</li><li>Deb, C., Lafrance-Corey, R. G., Zoecklein, L., Papke, L., Rodriguez, M., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demyelinated+axons+and+motor+function+are+protected+by+genetic+deletion+of+perforin+in+a+mouse+model+of+multiple+sclerosis.">Demyelinated axons and motor function are protected by genetic deletion of perforin in a mouse model of multiple sclerosis.</a> J. Neuropath. Exp. Neurol. 68, 1037-1048 (2009).</li><li>Deb, C., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKG2D+contributes+to+efficient+clearance+of+picornavirus+from+the+acutely+infected+murine+brain.">NKG2D contributes to efficient clearance of picornavirus from the acutely infected murine brain.</a> J. Neurovirol. 14, 261-266 (2008).</li><li>Donovan, J., &amp;amp, P. . B. r. o. w. n., , P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Euthanasia.">Euthanasia.</a> Current Protocols in Neuroscience. , A.4H.1-A.4H.4 (2005).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Handling+&#38;+restraint.">Handling &#38; restraint.</a> Current Protocols in Immunology. 73, 1.3.1-1.3.6 (2006).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parenteral+injections.">Parenteral injections.</a> Current Protocols in Neuroscience. , A.4F.1-A.4F.9 (2005).</li><li>Holmes, K. L., Otten, G., Yokoyama, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+analysis+using+Becton+Dickinson+FACS+Calibur.">Flow cytometry analysis using Becton Dickinson FACS Calibur.</a> Current Protocols in Immunology. , 5.4.1-5.4.22 (2002).</li><li>Howe, C. L., Ure, D., Adelson, J. D., LaFrance-Corey, R., Johnson, A., Rodriguez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+cells+directed+against+a+viral+peptide+contribute+to+loss+of+motor+function+by+disrupting+axonal+transport+in+a+viral+model+of+fulminant+demyelination.">CD8+ T cells directed against a viral peptide contribute to loss of motor function by disrupting axonal transport in a viral model of fulminant demyelination.</a> J Neuroimmunol. 188, 13-21 (2007).</li><li>Lipton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theiler's+virus+infection+in+mice:+An+unusual+biphasic+disease+process+leading+to+demyelination.">Theiler's virus infection in mice: An unusual biphasic disease process leading to demyelination.</a> Infect Immun. 11, 1147-1155 (1975).</li></ol>

我们经常使用流式细胞仪检测，以确定两者的大脑浸润细胞制备的质量，并区分不同人群的免疫细胞2，9 。急性发作的时间点，我们BILs方法产生炎症单核细胞内CD45hi人口以及CD45lo人口中的巨噬细胞的高百分比的高比例。这表明，在大脑中重现的免疫反应，我们的方法可以有力特点。 这种技术是特别重要的实验，需要一个良好的特点，从小鼠大脑中的免疫细胞的人口。渗透到宿主小鼠经尾静脉注射7的白细胞注入了我们的大脑的，我们曾进行过继转移实验，我们已 在体外实验中9通过各种特点的BILs。我们的BILs准备，必须区分不同人群的居民的脑细胞，包括居民的巨噬细胞和小胶质细胞的免疫细胞的实验，特别是在有利的。利益的另一种应用，包括实时RT - PCR技术从BILs在感染后7天隔离识别效应功能3的总RNA进行分析。例如，我们测量GAPDH的，颗粒酶B和穿孔RNA在感染后7天收集穿孔，主管和缺陷动物感染与TMEV BILs。我们还利用我们BILs的方法来确定如何NKG2D的结算TMEV从急性感染小鼠 4的大脑。 的方法有几个重要方面。重要的是把所有的解决方案室温事先准备，以避免不必要的压力的白细胞，并确保适当的光密度分离。对白细胞的压力会导致细胞死亡，这使得它们尤其是不可靠的居住人口的细胞是过继转移到宿主动物体内实验。我们还决定，不仅改变使用冷Percoll不连续梯度密度，而且也影响白细胞的质量。另一个关键因素是足够的和温和的同质化脑组织。我们已经发现，在显微镜和流式细胞仪所看到的细胞碎片丰富的脑组织结果的不足和粗糙的同质化。细胞碎片也可能会导致不使劲用适当大小的细胞过滤器的脑组织匀浆。适当注重细节是，在进一步的实验和分析准备充分和足够的的BILs的关键。

<table border="1"><tbody><tr><th>试剂</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> TMEV </td><td>豪实验室</td><td> N / A </td><td></td></tr><tr><td>丹尼尔的应变</td><td> Invitrogen公司</td><td> 21063-029 </td><td></td></tr><tr><td>异氟醚</td><td> Novaplus </td><td> NDC 0409-3292-49 </td><td></td></tr><tr><td>橡树岭圆底离心管</td><td> NALGENE </td><td> 3118-0030 </td><td></td></tr><tr><td> RPMI 1640培养</td><td> Invitrogen公司</td><td> 11875-093 </td><td></td></tr><tr><td> Percoll </td><td> GE医疗集团</td><td> 17-0891-02 </td><td></td></tr><tr><td> 10X PBS </td><td>罗氏公司</td><td> 11666789001 </td><td></td></tr><tr><td>聚蔗糖 - 帕确加</td><td> GE医疗集团</td><td> 17-1440-02 </td><td></td></tr><tr><td>台盼蓝0.4％（W / V） </td><td> Mediatech </td><td> 25 - 900 - CI </td><td></td></tr><tr><td> 15 ml锥形管</td><td>屋宇署猎鹰</td><td> 352097 </td><td></td></tr><tr><td> 7 mL玻璃耐热品牌Tenbroeck组织磨床</td><td>费舍尔</td><td> 08 - 414 - 10B </td><td></td></tr><tr><td> 40微米的细胞过滤器</td><td>屋宇署猎鹰</td><td> 352340 </td><td></td></tr><tr><td> 50 mL锥形</td><td>屋宇署猎鹰</td><td> 352070 </td><td></td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A9647 </td><td></td></tr><tr><td>叠氮化钠</td><td>西格玛</td><td> S8032 </td><td></td></tr><tr><td> CMF - PBS </td><td> Mediatech </td><td> 21 - 040 - CV </td><td></td></tr><tr><td>流式细胞仪管</td><td>屋宇署猎鹰</td><td> 352054 </td><td></td></tr><tr><td>胎牛血清</td><td>西格玛</td><td> F4135 </td><td></td></tr><tr><td> 2.4G2杂交瘤细胞</td><td> ATCC </td><td> HB - 197 </td><td></td></tr><tr><td>合演V型底板</td><td>康宁</td><td> 3894 </td><td></td></tr><tr><td> Allegra的X - 22R离心机或同等学历</td><td>贝克曼</td><td> N / A </td><td></td></tr><tr><td> 96孔板桶和转子</td><td>贝克曼</td><td> S2096 </td><td></td></tr><tr><td>固定角转子</td><td>贝克曼</td><td> F0360 </td><td></td></tr><tr><td>多聚甲醛</td><td>西格玛</td><td> P6148 </td><td></td></tr><tr><td> BD流式细胞仪刀魂</td><td> BD公司</td><td> N / A </td><td></td></tr><tr><td> FlowJo 7.5 </td><td>树星公司</td><td> N / A </td><td></td></tr><tr><td> CD45 </td><td> BD公司</td><td> 557235 </td><td>克隆:30 - F11 </td></tr><tr><td> GR1（Ly6C / G） </td><td> BD公司</td><td> 553128 </td><td>克隆:RB6 - 8C5 </td></tr><tr><td> CD11b的</td><td> ebiosciences </td><td> 17-0112-83 </td><td>克隆:M1/70 </td></tr><tr><td> F4/80 </td><td> ebiosciences </td><td> 17-4801-82 </td><td>克隆:BM8 </td></tr></tbody></table>

isolation of brain-infiltrating leukocytes

我们描述了一个准备从老鼠的大脑浸润白细胞（BILs）的方法。我们演示了如何Theiler鼠脑脊髓炎病毒（TMEV）通过一个快速的颅内注射技术和如何净化的全脑细胞浸润的白细胞富集人口感染小鼠。简单地说，小鼠是在一个封闭的腔异氟醚麻醉徒手与汉密尔顿注射器注入额叶皮层。然后被杀害后，小鼠感染的异氟醚过量和整个大脑中提取的RPMI匀浆与Tenbroeck组织磨床，在不同的时间。通过连续的30％Percoll梯度离心脑组织匀浆，以去除髓鞘和其他细胞碎片。细胞悬液，然后是紧张在40微米，洗净后，一个不连续的聚蔗糖 - 帕确加梯度离心选择和净化的白细胞。的白细胞，然后洗净，并在适当的缓冲区，通过流式细胞仪检测免疫重悬于。流式细胞仪检测显示人口先天免疫细胞的C57BL / 6小鼠在感染的早期阶段。多个亚群的免疫细胞在感染后24小时，目前，在BILs GR1 +，CD11b的+和F4/80 +细胞的丰富人口。因此，这种方法是有用的表征在大脑中的急性感染的免疫反应。

Watch this Scientific Journal Video about Isolation of Brain-infiltrating Leukocytes at JoVE.com

Isolation of Brain-infiltrating Leukocytes

一个快速的方法来获得从小鼠脑组织浸润的白细胞描述。这种方法利用了连续Percoll梯度和不连续的聚蔗糖梯度选择和净化白细胞富集层。然后可能会被隔离白细胞流式细胞仪测量的特点。

脑浸润白细胞的分离

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

isolation-of-brain-infiltrating-leukocytes

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

19.0K 次观看.  Mayo Clinic College of Medicine. 一个快速的方法来获得从小鼠脑组织浸润的白细胞描述。这种方法利用了连续Percoll梯度和不连续的聚蔗糖梯度选择和净化白细胞富集层。然后可能会被隔离白细胞流式细胞仪测量的特点。

视频: Isolation of Brain-infiltrating Leukocytes

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

心脏功能免疫细胞的分离

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

科研

免疫与感染

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

脑幽静的白细胞的分离与分析<em&gt;伯氏疟原虫</emANKA感染的小鼠

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

脂肪组织免疫细胞的分离

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

生物能量和氧化突发:协议用于人类白细胞和血小板的隔离与评价

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

对白细胞从流程的分析招聘基质细胞的影响

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

从人类母胎界面分离白细胞

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

从小鼠组织在母胎界面分离白细胞

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating肺白细胞的选择性收获

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating肝白细胞的选择性收获

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...