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，用小剪刀剪尾巴尖（约1毫米）。另外，一个小尾巴，可以使用25 G针的穿刺。 </li><li>从鼠标的尾部静脉接近尾声的磨砂年底的显微镜载片上，将一滴血。 </li><li>成45°角放置一个第二滑动（扩频器）和备份到的血液的液滴。一旦血液中的沿边缘扩散，推动的吊具滑前夕NLY对面的血涂片的显微镜幻灯片。让涂抹在空气中干燥。 </li><li>在100％甲醇，持续30秒，然后修复血涂片染色在新鲜制备的10分钟的吉姆萨滑动。 </li><li> 1分钟，用流动的水冲洗血涂片，让空气干燥和审查，在显微镜下（100倍，浸油）的幻灯片。列举1000红细胞和猪红细胞内，计算百分比原虫。供体小鼠原虫水平应达到2.5-5％之间，才可以感染实验动物流血。 </li><li>采集血液从供体小鼠的眼窝出血，使用肝素的的微血细胞比容毛细管。所有的实验都符合沃尔特和伊丽莎·霍尔研究所动物伦理委员会的要求。根据当地动物伦理委员会要求的其他出血程序可能会用到。溶解所需量的血液，在RPMI培养基中的终浓度为1X10 6 pRBC/0.2毫升。想想看，正常小鼠血细胞比容是〜6×10 9的红血细胞/ 1毫升。 </li><li>感染实验的C57BL / 6小鼠（8-12周龄），通过注入腹膜内（ip）1×10 6 pRBC/0.2毫升。 </li><li>监控原虫感染小鼠，请按照下列步骤1.3-1.8。原虫血症应确定每2-3天开始第2天或3 PI </li><li>在C57BL / 6小鼠会表现为驼背的外观，严重的疟疾发病的时候总是毛皮和低活性。有些鼠标可以恢复在这个阶段，但发展自翻正反射损失表示不可逆的疾病和动物必须实施安乐死。 </li></ol> 2。心内灌注小鼠脑提取<ol><li>通过固定的500毫升水库60厘米柱支架的顶部连接到一列保持架组装手动重力灌注系统。将塑料管的底端水库，确保油管钳，控制缓冲流。连接管连接到一个23 G针。费尔l对水库PBS（保持在22°C），打开钳和缓冲运行通过管道，以除去所有的气泡。 </li><li>安乐死P.伯氏疟原虫 ANKA的CO 2吸入受感染的小鼠。踏板的情况下，轨道和呼吸反应，确认死亡。 </li><li>针安乐死鼠标背部上的一个塑料夹层板包含在一个塑料托盘的后腿和前爪。根据当地动物伦理委员会的要求麻醉，可管理的前心内pefusion开始。 </li><li>用70％乙醇擦拭腹侧。 </li><li>用大剪刀和镊子，打开沿正中线皮肤暴露胸腔。折叠和引脚的皮肤向两侧。 </li><li>用细镊子和保持胸骨切开膈肌和切断胸骨两侧的肋骨。 ，请注意不要损坏任何大血管。 </li><li>引脚的肋骨，胸骨松散的头。 </li><li>保持心室机智Ĥ细钳和右心房用细剪刀小心地切割。 </li><li>插入23G针连接到重力灌注系统进入PBS被运行时朝向升主动脉左心室。插入只有0.5厘米的针尖。 </li><li>灌注鼠标为5分钟或直到出水是明确的。 </li><li>取消固定鼠标，并把它放到它的腹部。用70％乙醇擦拭头。 </li><li>用大剪刀，作一个切口正上方的脊髓型颈椎病领域。 </li><li>用细剪刀，中位数尾部的喙切在颅底和剥离皮肤，以露出头骨。 </li><li>握住头部，将一个小的剪刀的刀片的到每个轨道空腔和轨道之间作一个切口。 </li><li>做一个纵向切割沿矢状缝，小心剥离头骨上每个大脑半球向外。 </li><li>用锅铲轻轻抬起大脑，将其放入10 mL离心管中含有RPMI培养基。 </li></ol>3。分离脑幽静的白细胞（BSL） <ol><li>工作在一个安全柜，地点的新鲜收获的脑含有3-5毫升的RPMI组织培养基的皮氏培养皿中的不锈钢的细胞过滤网（40-60筛目大小）的顶部。 </li><li>脑组织切小块。 </li><li>将脑组织的小片，通过细胞过滤网使用晶体柱塞。 </li><li>脑匀浆转移到10毫升管中，离心，在250×g离心10分钟，在4℃下</li><li>溶解沉淀，3毫升的RPMI培养基中，含有0.05％胶原酶D和一2U/ml DNA酶</li><li>旋转混合物在室温下的30分钟。通过按压该混合物通过70微米尼龙细胞过滤和/或通过在冰上孵育5分钟，除去细胞碎片。 </li><li>到7毫升30％的Percoll坐垫和离心机在400×g离心20分钟，在室温下（没有刹车）上铺脑匀浆。 </li><li>沉淀重悬在1ml红细胞裂解缓冲液，在冰上孵育5分钟，以裂解贴壁PRBC，心内灌注，不能从大脑中删除。 </li><li>加入9毫升RPMI培养基，洗涤细胞并离心，在250×g离心10分钟，在4℃下</li><li>重悬BSL含颗粒在50-100微升RPMI培养基中和到Eppendorf管中的传输单元。 </li><li>稀释的细胞样品为5-10微升等分1:2的台盼蓝活细胞识别。 </li><li>计数存活的细胞，在显微镜下用血细胞计数。从第6天PI起，当P.伯氏疟原虫的 ANKA感染小鼠显示神经系统体征，，20,000-100,000楼宇安全贷款计划可以恢复。 </li></ol> 4。多色流式细胞术免疫分型BSL <ol><li>离心细胞在250×g离心10分钟，在4℃，吸出上清和重悬沉淀在50μl的染色缓冲液（PBS，1％胎牛血清（FCS），EDTA2毫米）中。 </li><li>加入1微升纯化anti-CD16/32的抗体（Fcblock）。 </li><li>培养细胞10分钟在冰。 </li><li>用1毫升的染色缓冲液洗细胞。细胞在250×g离心10分钟，在4℃下离心，吸出上清液。 </li><li>在50μl的染色缓冲液含有预先确定的最佳稀释所需的荧光抗体， 即抗-CD4，抗-CD8，抗-TCR和抗NK1.1重悬细胞。 </li><li>在冰上孵育1小时。 </li><li>用1毫升的染色缓冲液洗细胞。细胞在250×g离心10分钟，在4℃下离心，吸出上清液。 </li><li>重悬在100μl的PBS中的细胞。 </li><li>转移细胞的流式细胞术的塑料管。 </li><li>使用流式细胞仪，获取至少5,000-10,000事件。应包括适当的未染色的细胞对照组和荧光补偿样品的要求。 </li><li>使用适当的流式细胞仪软件分析结果。 </li></ol>

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没有利益冲突的声明。

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. ...

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JoVE Journal

Immunology and Infection

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

12.7K 次观看.  The Walter and Eliza Hall Institute of Medical Research. 贴壁脑血管的炎性白细胞隔离的方法伯氏疟原虫 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。

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

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

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 ...

科研

免疫与感染

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

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

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

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫</i

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

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 ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

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

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

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

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

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-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 ...