Name: isolation and analysis of brain-sequestered leukocytes from plasmodium berghei anka-infected mice
Uploaded: 2013-01-02T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice at JoVE.com

作者想感谢吴利亚纳马茨凯维奇技术援助。这项工作可以通过维多利亚州政府运营基础设施的支持，澳大利亚政府国家健康与医学研究委员会IRIISS和项目资助1031212。

1。感染小鼠的P.伯氏疟原虫的ANKA <ol><li>除霜取冻存P.伯氏疟原虫 ANKA PRBC。 </li><li>抑制的脑疟疾抗BALB / c小鼠供体小鼠（8-12周龄）使用双手的约束技术。 100-200微升猪红细胞使用一个1毫升的胰岛素注射器（28G针）注入小鼠。常规1-2个供体小鼠注射。 </li><li>在天4-5感染后（PI）从笼中取出供体小鼠，并将其放置在工作站或一次性垫。 </li><li>轻轻地抑制小鼠的尾巴末端，用小剪刀剪尾?...

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BSL的分离和分析的方法，它允许迁移到大脑响应于在实验小鼠模型中的组织损伤或感染的炎症细胞的定性和定量。的心脏内灌注器官提取前的血液舱和随后的细胞隔离步骤，用于除去的引入与非炎症性的循环中的白细胞的炎症细胞是有用的，以防止污染。鼠脑脊髓炎病毒等炎性细胞渗入脑实质5嗜神经性感染，这可能不是一个基本要求，但在啮齿类动物疟疾，其中特别是有关保持隔离受感...

<table border="1"><tbody><tr><td> 的试剂的名称 </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> 解决方案和缓冲区 </td></tr><tr><td>姬姆萨的海岸伊红美蓝溶液</td><td>默克密理博</td><td> 1.09204.0500 </td><td>在蒸馏水中的1:10稀释</td></tr><tr><td> RPMI培养基</td><td></td><td></td><td>鼠标张力</td></tr><tr><td>鼠标张力PBS </td><td></td><td></td><td>的20mM磷酸钠，0.149的NaCl，pH 7.3的</td></tr><tr><td> 0.4％的台盼蓝</td><td> Sigma Aldrich公司</td><td> T-8154 </td><td> 1:2稀释</td></tr><tr><td>胶原酶ð </td><td>沃辛顿生化</td><td></td><td></td></tr><tr><td> Deoxyribonu易洁卫浴（DNA酶）I </td><td> Sigma Aldrich公司</td><td> D4263-5VL </td><td>从牛胰腺</td></tr><tr><td>珀</td><td> GE医疗集团</td><td> 17-0891-01 </td><td>在PBS中的30％溶液</td></tr><tr><td>高纯三</td><td> Invitrogen公司</td><td> 15505-020 </td><td></td></tr><tr><td>氯化铵（NH 4 Cl的 ） </td><td>高纯度化学剂</td><td> 10017 </td><td></td></tr><tr><td>红色细胞裂解液</td><td></td><td></td><td> 17毫摩尔Tris，14NM，pH值7.2 NH 4 Cl的 </td></tr><tr><td> FCS </td><td> Gibco公司</td><td> 1009 </td><td></td></tr><tr><td> EDTA二钠盐</td><td>默克</td><td> 10093.5V </td><td> 0.1M，pH 7.2的</td></tr><tr><td></td><td></td><td></td><td> 抗体和结合物 </td></tr><tr><td> ANTI-MOUSE CD16/CD32（FC块），克隆2.4G2 </td><td> BD Pharmingen公司</td> &lt;TD&gt; 553142 </td><td> 1微升在50μl的染色缓冲液（0.5mg/50毫升） </td></tr><tr><td> FITC-抗小鼠CD4，克隆H129.19 </td><td> BD Pharmingen公司</td><td> 553651 </td><td></td></tr><tr><td> PE-抗小鼠NK1.1，克隆PK136 </td><td> BD Pharmingen公司</td><td> 553165 </td><td></td></tr><tr><td> PerCPCy5.5-抗小鼠CD8，克隆53-6-7 </td><td> BD Pharmingen公司</td><td> 551162 </td><td></td></tr><tr><td> APC-抗小鼠TCR-β，克隆H57-597 </td><td> BD Pharmingen公司</td><td> 553174 </td><td></td></tr><tr><td> PE抗小鼠CXCR3，克隆220803 </td><td> R＆D Systems公司</td><td> FAB1685P </td><td></td></tr><tr><td>生物素化的抗小鼠CCR5，克隆C34-3448 </td><td> BD Pharmingen公司</td><td> 559922 </td><td></td></tr><tr><td> Steptavidin-PerCP-Cy5.5标记</td><td> BD Pharmingen公司</td><td> 551419 </td><td></td></tr><tr><td></td><td></td><td></td><td> 设备和材料 </td></tr><tr><td>的Superfrost显微镜幻灯片</td><td>博士伦门泽尔 - 格拉泽</td><td></td><td></td></tr><tr><td>解剖镊子，剪刀</td><td>香港地产建设商会INSTRUMENTE </td><td></td><td></td></tr><tr><td> 500毫升PBS水库</td><td>的Nalgene </td><td></td><td></td></tr><tr><td>橡胶管</td><td></td><td></td><td></td></tr><tr><td> 23G针</td><td> BD PrecisionGlide </td><td> 302008 </td><td></td></tr><tr><td>细胞分离试剂盒含有金属筛</td><td> Sigma Aldrich公司</td><td> CD-1 </td><td></td></tr><tr><td> 70μm的尼龙细胞过滤器</td><td> BD猎鹰</td><td> 352350 </td><td></td></tr><tr><td>血球计数仪</td><td>有限公司纽鲍尔</td><td> 717810 </td><td></td></tr><tr><td>流式细胞仪检测管</td><td>乙ð猎鹰</td><td> 352008 </td><td></td></tr></tbody></table>

isolation and analysis of brain-sequestered leukocytes from plasmodium berghei anka-infected mice

我们描述的方法脑血管P.贴壁炎症细胞的分离和鉴定伯氏疟原虫 ANKA感染的小鼠。感染这种寄生虫的应变结果在诱导实验性脑疟疾，神经系统综合征的易感小鼠株，概括的某些重要方面介导的恶性疟原虫在人类1,2严重的疟疾。成熟形式的血液阶段疟疾感染的红细胞的表面上，这允许它们绑定到血管内皮细胞表达寄生蛋白质。这个过程导致的阻塞血液流动，导致缺氧，出血3，，也刺激炎性白细胞招聘网站的寄生虫封存。 不像其他的感染，即neutrotopic的病毒4-6，两个疟疾寄生的红血细胞（PRBC）以及相关联的inflammatory白细胞始终处于固定状态，而不是浸润脑实质内的血管。因此，为了避免污染螯合白细胞与非炎症性的循环细胞，广泛感染小鼠心脏内灌注器官提取和组织处理之前在此过程中是必需的，以除去血液舱。灌注后，大脑的收获和解剖小块。的组织结构进一步破坏胶原酶和DNA酶一酶处理由此产生的脑组织匀浆，然后离心分离上的Percoll梯度使脑幽静的白细胞分离（BSL）的髓鞘和其​​他组织碎片。计数中分离出细胞，然后洗净，用血球计数仪和荧光抗体染色后用流式细胞仪分析。 这个程序允许全面的表型特征的炎性白细胞迁移到大脑中重新响应各种刺激，包括中风，以及病毒或寄生虫感染。该方法还提供了一个有用的工具，用于评估新型的消炎治疗，在临床前动物模型。

图中的结果。 2显示比例和绝对数量不同的BSL人口的的灌流或unperfused疟疾病毒感染者和天真的控制小鼠的大脑恢复。隔离BSL议定书文本中所示，用PE-抗NK1.1和APC-抗-TCR-β抗体染色。与以前的研究结果7-9，αβTCR+ T细胞组成一个BSL池灌注感染疟疾的小鼠（第6天PI），大脑中的高比例。这个人口似乎是显着的人数不足非灌流动物大脑中（图2A-B）。具有相当高的百分比和双阴性细胞的总数（αβTCR ...

Watch this Scientific Journal Video about Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice at JoVE.com

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

贴壁脑血管的炎性白细胞隔离的方法<em&gt;伯氏疟原虫</em&gt; ANKA感染的小鼠进行说明。可以量化的方法，以及后的孤立白细胞表型特征流式细胞仪荧光抗体染色和随后的分析。

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. ...

Research

JoVE Journal

Immunology and Infection

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

Isolation of Brain-infiltrating Leukocytes

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 ...

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An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

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Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

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

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

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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 ...

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Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

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从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活<em&gt;疟原虫</em&gt;孢子

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

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 ...