这项工作是由一个UNCF默克研究生学研究论文奖学金（JLM），国家心脏，肺，血液研究所授予RO1 - HL - 62228（JSJ）和RO1的HL - 073990（JSJ）的支持。

1。汉克斯“平衡盐溶液的制备（HBSS） <ol><li> 1000毫升高压灭菌器1升玻璃烧杯，量筒，搅拌棒。 </li><li>重组蒸压1 L玻璃烧杯用双蒸水900毫升标记的HBSS粉末的解决方案。 </li><li> HEPES钠+盐3.4克的HBSS混合溶液和pH值调整到7.35-7.41之间的价值，无论是使用浓盐酸或氢氧化钠。 </li><li>一旦所需的pH值已达到并保持稳定，加5克牛血清白蛋白在室温下的解决方案，直到完全溶解白蛋白。 </li><li>添加的抗生素，antimyotic，解决方案和双蒸馏水10毫升，直至总量在1000毫升标记。 </li><li>搅拌彻底的解决方案。 </li><li>过滤器的HBSS解决方案，并分成50毫升猎鹰管，供以后使用。其他管可以被冻结，并在以后的日子使用。 </li></ol> 2。动物PREPARATION - 插入气管套管<ol><li>准备一盘插入气管套管（即镊子，剪刀，气管插管，3-0黑色编织缝合，小动物呼吸机等）所需的仪器和材料。 </li><li>一旦大鼠氯胺酮/甲苯​​噻嗪的鸡尾酒在2氯胺酮1甲苯噻嗪的比例是完全麻醉，刮胡子，大鼠腹部，胸部和喉咙。为了确保已经达到深麻醉状态，捏老鼠的脚趾，检查踏板肢体撤回。确保大鼠深麻醉前使所有的切口。 </li><li>将其头部大鼠砧板上提出的，是在30度角斜</li><li>从中期腹部下颌下方做一个皮肤切口。解剖走的颈部，暴露气管前壁三角形的肌肉。 </li><li>下放置3-0黑色编织缝合气管。 </li><li>在气管软骨环之间的一个切口ð插入气管套管。领带的缝合紧密，以确保气管插管气管。 </li><li>气管插管连接小动物呼吸机。 </li></ol> 3。免疫细胞分离仪器的准备工作<ol><li>准备一个托盘，与免疫细胞分离过程中的工具（如镊子，剪刀，HBSS中，修剪过的针快速导管软聚四氟乙烯24号巴克斯特血管） </li><li>准备两个10CC注射器，引入心包囊和储备的另一种缓冲吸HBSS中含有细胞与HBSS中填充一个注射器。 3.2.1减少其分离过程中的原始长度的1 / 2至3 / 4的24号巴克斯特血管聚四氟乙烯以上的快速导管针（1.6厘米）提示。 </li><li>将所有注射器，缓冲区和一个空的15毫升猎鹰管（保存过程完成后，提取液）在冰上。 </li></ol> &lt;P类=“j​​ove_title”&gt; 4。免疫细胞分离<ol><li>请在腹壁沿白线正中切口，直到你达到剑突。 </li><li>开始略有横向至剑突过程，通过双边切口肋骨笼继续朝腋下的角度。要小心，不要刺穿肺部，心脏或心包。 </li><li>一旦胸骨两侧的切口完成后，解剖远离较低的肋骨膜片，切割外侧到内侧。 注意：永远知道心包连接到膈肌，胸骨，以避免损坏。作为一个具有里程碑意义的的镰状韧带，要注意的是对面的隔膜附着心包。此外，如果被切断，心包分离细胞的能力都将丢失。 </li><li>轻轻抬起一侧胸腔，暴露胸腔和心脏（心包封装）。</li><li>以HBSS中充满注射器和心包囊中找到一个点之间的胸骨和心脏（最好超过尾鳍延髓）约一半的方式，插入聚四氟乙烯尖的快速通过两层心包导管缩短。 </li><li>导管到位后，轻轻地进行填充2至3毫升HBBS缓冲区心包空间。你会立即知道，当您发现心包空间不断扩大，如果导管妥善​​安置。如果没有填充的见证下，撤销和重新定位的快速导管。 </li><li>心包囊内充满时，其他10毫升注射器和地点提示心包，并开始吸缓冲区。可以使用相同的项目，洞，或其他可以创造的。确保针尖不来太接近，特别是在心脏底部心包囊的墙壁上，同时提供吸力，。这可能会导致穿刺心包囊和细胞含有细胞的损失第</li><li>在吸缓冲区，可移动导管尖端的心包囊内左右，以收集尽可能多的缓冲区。 </li></ol> 注意：如果你这样做轻微撕裂心包，使心脏比原来的洞大顶部的孔，不要惊慌。只要心包仍然包围着的心脏的话，你仍然可以执行另一场心脏，尽管有少量的缓冲洗净。收集细胞的最优数量，需要三至四个额外的清洗。 <ol start="9"><li>将猎鹰管置于冰上提取的缓冲区。 </li><li>取出心脏，肺，和/或任何其他组织如果需要作进一步的研究。大鼠免疫细胞和其他组织的集合后，必须迅速和人道安乐死。 </li><li> 10分钟200克在4 ° C离心收集的免疫细胞样本始终保持在冰上管在不使用时。 </li><li>删除在1毫升Hyclone公司缓冲区或HBSS上清和重组颗粒。 </li></ol> 注：重要的是要计算每个心脏后，免疫细胞的分离过程中获得的免疫细胞的数量。 5。代表性的成果： 与这项技术，并与甲苯胺蓝染色和番红O /阿尔辛蓝获得一个孤立的心脏肥大细胞的例子是，分别在图2a和2b。可以看到在图3中，心脏肥大，维护他们的能力，释放组胺治疗后如复方48/80和P物质，如摩根等著名激活 。9代表散点图从流式细胞仪的CD4 +细胞CD8 +细胞，分别在图4和5。 T淋巴细胞是进一步查明并作为这项技术的细胞群phenotyped。 图1。心脏的免疫细胞的分离过程的流程图。 <img alt="图2" src="/files/ftp_upload/3020/3020fig2.jpg" /> 图2心脏肥大细胞的组织的代表图像染色如下：（一）与甲苯胺蓝和（B）是番红O /阿尔辛蓝。 <img alt="图3" src="/files/ftp_upload/3020/3020fig3.jpg" /> 图3从心外膜心肌地区隔离，并与化合物48/80（10μg/mL）和P物质（10μM）刺激心脏肥大细胞的功能研究。 <img alt="图4" src="/files/ftp_upload/3020/3020fig4.jpg" /> 代表孤立的CD4 +细胞与P物质（1:100 dilut标记流式细胞仪散点图图4。离子）。 <img alt="图5" src="/files/ftp_upload/3020/3020fig5.jpg" /> 图5。孤立的CD8 +标记细胞与P物质（1:100稀释）代表流式细胞仪。

<ol>
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我们什么都没有透露。

这种隔离心脏免疫细胞的方法，使无不需要激活，通常发生在传统的酶消化法分离技术，分析其功能，表型，成熟。这种技术获得的平均细胞总数约400000元大鼠心脏，差这些细胞的染色和流式细胞仪分析表明（〜70％），巨噬细胞（〜12％）和肥大细胞，T淋巴细胞混合的人口使用了这种技术。（〜12％）。13，14因此，心脏的免疫细胞群的进一步分离纯化方法是必要的。隔离的免疫细胞，可...

<table border="1"><tbody><tr><td>试剂和设备名称</td><td>公司</td><td>目录编号</td><td>评论</td></tr><tr><td>汉克斯“平衡盐</td><td>西格玛</td><td> H4891 </td><td>一瓶1 L汉克斯&#39;平衡盐 解决方案。重组汉克斯&#39;平衡盐 解决方案作为一个过程的一部分。 </td></tr><tr><td>抗生素的Antimyotic </td><td> Gibco公司</td><td> 15240-062 </td><td></td></tr><tr><td> HEPES </td><td>西格玛</td><td> H3784 </td><td></td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A3912 </td><td></td></tr><tr><td> 250毫升Nalogene滤瓶</td><td> Thermoscientific </td><td> 157-0020 </td><td></td></tr><tr><td> 12 N盐酸</td><td> Fisher Scientific则</td><td>中科院7647-01-0 </td><td>对于6个N，添加盐酸等量去离子水。 </td></tr><tr><td> 10 N的氢氧化钠</td><td> Fisher Scientific则</td><td> SC267 </td><td> 5 N，添加去离子水相等体积的NaOH。 </td></tr><tr><td>双蒸水</td><td></td><td></td><td></td></tr></tbody></table>具体设备表： <table border="1"><tbody><tr><td>试剂和设备名称</td><td>公司</td><td>目录编号</td><td>评论</td></tr><tr><td> - 3 - O缝合丝绸编织</td><td>爱惜康产品</td><td> SA64H </td><td></td></tr><tr><td>小动物呼吸机</td><td> CWE公司</td><td> SAR - 830 </td><td>设置呼吸频率每分钟40.5次呼吸。 </td></tr><tr><td>斜角18号针头</td><td> BD公司</td><td> 305199 </td><td>修剪其原始长度的1 / 2至3 / 4使用气管套管针。 </td></tr><tr><td>聚四氟乙烯提示24计巴克斯特快速导管导管（带针头删除） </td><td>巴克斯特</td><td> 2N111 </td><td>聚四氟乙烯提示长度减少1 / 2至3 / 4。 </td></tr><tr><td> 10毫升注射器</td><td> Fisher Scientific则</td><td> 14 - 823 - 16B </td><td></td></tr><tr><td> 15毫升猎鹰管</td><td> BD公司</td><td> 352097 </td><td></td></tr><tr><td> Sorvall RT6000B离心机</td><td> Fisher Scientific则</td><td> 75-004-377 </td><td>记住预冷，并保持在4 ° C。 </td></tr><tr><td> Hyclone公司</td><td> Thermoscientific </td><td> S300030.02 </td><td></td></tr><tr><td>血球</td><td> Hausser科学</td><td> HS - LB - 1483 </td><td></td></tr></tbody></table>

isolation of functional cardiac immune cells

心脏免疫细胞获得利益的角色，他们在许多心脏疾病的病理重塑发挥1-5这些免疫细胞，其中包括肥大细胞，T细胞和巨噬细胞;存储和释放的多种生物活性介质，包括细胞因子和蛋白酶（如胰蛋白酶）。6-8这些调解员已被证明是通过激活基质金属蛋白酶或胶原蛋白的积累引起心脏成纤维细胞的功能，通过调节细胞外基质代谢的关键球员。9-11然而，隔离心脏免疫细胞技术一直是个问题，因为他们使用细菌胶原酶消化心肌组织。这种技术的原因，从而激活免疫细胞功能的损失。例如，心脏肥大细胞变得大大减少响应的化合物，导致脱粒， 因此 ，我们开发出一种技术，允许对T他隔离心脏功能的免疫细胞，这将导致更好地了解了这些细胞在心脏疾病中的作用。 13，14 此方法需要一个熟悉大鼠剑突，腋下和镰状韧带的解剖位置，对心脏和心包。这些标志性建筑是重要的，以提高手术成功和心脏的免疫细胞，以确保较高的产量。这些孤立的心脏，免疫细胞可以用于表征功能，表型，成熟，并与其他心肌细胞共培养实验，以获得一个更好地了解它们之间的相互作用。

Watch this Scientific Journal Video about Isolation of Functional Cardiac Immune Cells at JoVE.com

Isolation of Functional Cardiac Immune Cells

这种隔离的心脏功能的免疫细胞的方法提供了一种替代传统的方法胶原酶消化，从而导致不​​必要的免疫细胞活化，从而导致了这些细胞的减少响应。我们隔离的方法，产生心脏功能的免疫细胞，避免与酶消化相关的问题。

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 ...

isolation-of-functional-cardiac-immune-cells

Research

JoVE Journal

Immunology and Infection

14.9K 次观看.  University of South Carolina- School of Medicine. 这种隔离的心脏功能的免疫细胞的方法提供了一种替代传统的方法胶原酶消化，从而导致不​​必要的免疫细胞活化，从而导致了这些细胞的减少响应。我们隔离的方法，产生心脏功能的免疫细胞，避免与酶消化相关的问题。

视频: Isolation of Functional Cardiac Immune Cells

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

科研

免疫与感染

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

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 ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

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 ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

分离，鉴定和牙龈免疫细胞网络的功能性考试

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

人雌性生殖道树突状细胞表型鉴定的分离及功能研究

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic immune response. The activation of MCs via the cross-linking of surface IgE bound to high affinity IgE receptor (Fc&#949;RI), as well as through the stimulation of several other receptors, initiates the rise of the free intracellular Ca2+ level ([Ca2+]i) that promotes the release of inflammatory and allergic mediators. The identification of molecular constituents involved in these signaling pathways is crucial for understanding the regulation of MC function. In this article, we describe a protocol for the isolation of murine connective tissue type MCs by peritoneal lavage and cultivation of peritoneal MCs (PMCs). Cultures of MCs from various knockout mouse models by this methodology represent a useful approach to the identification of proteins involved in MC signaling pathways. In addition, we also describe a protocol for single cell Fura-2 imaging as an important technique for the quantification of Ca2+ signaling in MCs. Fluorescence-based monitoring of [Ca2+]i is a reliable and commonly used approach to study Ca2+ signaling events, including store-operated calcium entry, which is of utmost importance for MC activation. For the analysis of MC degranulation, we describe a &#946;-hexosaminidase release assay. The amount of &#946;-hexosaminidase released into the culture medium is considered as a degranulation marker for all three different secretory subsets described in MCs. &#946;-hexosaminidase can easily be quantified by its reaction with a colorigenic substrate in a microtiter plate colorimetric assay. This highly reproducible technique is cost-effective and requires no specialized equipment. Overall, the provided protocol demonstrates a high yield of MCs expressing typical MC surface markers, displaying typical morphological and phenotypic features of MCs, and demonstrating highly reproducible responses to secretagogues in Ca2+-imaging and degranulation assays.

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Here, we present a protocol to isolate and cultivate murine peritoneal mast cells. We also describe two protocols for their functional characterization: a fluorescent imaging of intracellular free Ca2+ concentration and a degranulation assay based on colorimetric quantification of the released &#946;-hexosaminidase.

腹膜源性肥大细胞的分离及其功能特征的 Ca2 +成像和脱粒测定

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...