脑幽静的白细胞的分离与分析<em>伯氏疟原虫</emANKA感染的小鼠
Summary
贴壁脑血管的炎性白细胞隔离的方法<em>伯氏疟原虫</em> 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。
Abstract
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。
Protocol
1。感染小鼠的P.伯氏疟原虫的ANKA 除霜取冻存P.伯氏疟原虫 ANKA PRBC。 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。 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。 轻轻地抑制小鼠的尾巴末端，用小剪刀剪尾?…
Representative Results
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 …
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Solutions and buffers | |||
| Giemsa’s azur eosin methylene blue solution | Merck Millipore | 1.09204.0500 | 1:10 dilution in distilled water |
| RPMI medium | Mouse tonicity | ||
| Mouse tonicity PBS | 20 mM Sodium Phosphate, 0.149 NaCl, pH 7.3 | ||
| 0.4%Trypan Blue | Sigma Aldrich | T-8154 | 1:2 dilution |
| Collagenase D | Worthington Biochemical | ||
| Deoxyribonuclease (DNAse) I | Sigma Aldrich | D4263-5VL | From bovine pancreas |
| Percoll | GE Healthcare | 17-0891-01 | 30% solution in PBS |
| Ultrapure Tris | Invitrogen | 15505-020 | |
| Ammonium Chloride (NH4Cl) | AnalaR | 10017 | |
| Red Cell Lysis Buffer | 17 mM Tris,14 nM NH4Cl, pH 7.2 | ||
| FCS | Gibco | 1009 | |
| EDTA disodium salt | Merck | 10093.5V | 0.1M, pH 7.2 |
| Antibodies and conjugates | |||
| Anti-mouse CD16/CD32 (Fc Block), clone 2.4G2 | BD Pharmingen | 553142 | 1 μl in 50 μl staining buffer (0.5 mg/50 ml) |
| FITC-anti-mouse CD4, clone H129.19 | BD Pharmingen | 553651 | |
| PE-anti-mouse NK1.1, clone PK136 | BD Pharmingen | 553165 | |
| PerCPCy5.5-anti-mouse CD8, clone 53-6-7 | BD Pharmingen | 551162 | |
| APC-anti-mouse TCR-β, clone H57-597 | BD Pharmingen | 553174 | |
| PE-anti-mouse CXCR3, clone 220803 | R&D Systems | FAB1685P | |
| Biotinylated-anti-mouse CCR5, clone C34-3448 | BD Pharmingen | 559922 | |
| Steptavidin-PerCP-Cy5.5 | BD Pharmingen | 551419 | |
| Equipment and material | |||
| SuperFrost microscope slide | Lomb Menzel-Gläser | ||
| Dissection forceps, scissors | REDA Instrumente | ||
| 500 ml PBS reservoir | Nalgene | ||
| Rubber tubing | |||
| 23G needle | BD PrecisionGlide | 302008 | |
| Cell dissociation kit containing metal sieve | Sigma Aldrich | CD-1 | |
| 70 μm nylon cell strainer | BD Falcon | 352350 | |
| Hemocytometer | GmbH Neubauer | 717810 | |
| Flow cytometry tubes | BD Falcon | 352008 |
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Citer Cet Article
Ryg-Cornejo, V., Ioannidis, L. J., Hansen, D. S. Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice. J. Vis. Exp. (71), e50112, doi:10.3791/50112 (2013).