Name: isolation of brain-infiltrating leukocytes
Uploaded: 2011-06-13T12:00:00
Description: Watch this Scientific Journal Video about Isolation of Brain-infiltrating Leukocytes at JoVE.com

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?...

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	<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?...

<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 ...

Research

JoVE Journal

Immunology and Infection

Isolation of Functional Cardiac Immune Cells

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 of Mouse Lung Dendritic Cells

小鼠肺树突状细胞的分离

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

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

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

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. ...

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

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

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

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

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

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 ...