这项工作是支持部分由美国国立卫生研究院授予AI069358和BloodCenter研究基金会。

<ol><li>开始之前的过程中，下列项目需要收集和准备： <ul><li>冰</li><li> 27克针5ml注射器</li><li> 25克针5ml注射器</li><li>安装鼠标的泡沫塑料块和引脚</li><li>可以放在托盘上安装块</li><li>剪刀和镊子</li><li>收集管</li><li> 70％的乙醇</li><li> PBS与3％小牛血清（FCS）（前冷冻保存在冰） </li></ul></li><li>安乐死的鼠标，喷以70％乙醇和安装在它的后面的发泡胶块。 </li><li>使用剪刀和镊子切开腹膜外的皮肤，轻轻一拉，内层衬里的腹腔暴露。 </li><li>使用27克针进入腹膜腔注入5毫升冰冷的PBS（含3％FCS）。慢慢推针腹膜穿刺小心，不要被任何机关。 </li><li>注射后，轻轻按摩腹膜驱逐任何贴壁细胞的PBS液。 </li><li>插入一个25克的针，锥，附着在腹膜5 ml注射器和搜集流体移动时针尖轻轻地避免脂肪组织或其他器官堵塞。尽可能收集尽可能多的液体和存款取出后从注射器针管放在冰上收集细胞悬液。 可选：重复步骤4-6 </li><li>请在腹膜内的皮肤切口，并与钳皮肤而使用塑料的巴斯德吸管收集剩余的液体从腔。 </li><li>如果在步骤6或7可见血液污染检测，然后被污染的样品应该被丢弃。 </li><li>收集的细胞悬液在1500转的旋转，8分钟，弃去上清液，并计算所需的媒体或PBS重悬细胞。 </li></ol>替代协议获得硫乙醇酸盐引起巨噬细胞： 使用此方法，以获得更高的巨噬细胞的产量。 <ol><li> 3％（W / V）布鲁尔硫乙醇酸盐培养基（7）到每只小鼠腹腔注射5毫升。 </li><li>等待3-5天，并继续执行步骤1段收集细胞。 </li></ol> nonelicited方法相比，可以收集到约10倍多的巨噬细胞。唯一担心的是，此过程中获得的巨噬细胞，在他们的一些生理特性不同。 代表性的成果： 从unmanipulated鼠标，5-10万元的腹腔细胞，可以得到具有良好的隔离协议。在所有活细胞，B细胞的50-60％，〜30％的巨噬细胞和5-10％的T细胞（图1）。 <img src="/files/ftp_upload/1488/1488fig1.jpg" alt="图1" /> 图1。从腹膜腔中分离的细胞表型。以下隔离腹腔细胞，他们沾满抗鼠TCRβ- FITC，B220 - PE德州红，CD11b的太平洋蓝，CD23 - PE - Cy7和CD5 - APC。代表流式细胞仪图显示B细胞和T细胞（A），巨噬细胞（B），B1和B2的细胞（C）和阿维菌素B1a和B1B细胞（D）的百分比。

<ol>
	<li>Zhang, X., Goncalves, R., Mosser, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+and+characterization+of+murine+macrophages.">The isolation and characterization of murine macrophages.</a> Current Protocols in Immunology. , (2008).</li><li>Boes, M., Prodeus, A. P., Schmidt, T., Carroll, M. C., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+of+natural+immunoglobulin+M+in+immediate+defense+against+systemic+bacterial+infection.">A critical role of natural immunoglobulin M in immediate defense against systemic bacterial infection.</a> J. Exp. Med. 188, 2381-2386 (1998).</li><li>Mc Daniel, L. S., Benjamin, W. H., Forman, C., Briles, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+clearance+by+anti-phosphocholine+antibodies+as+a+mechanism+of+protection+in+experimental+pneumococcal+bacteremia.">Blood clearance by anti-phosphocholine antibodies as a mechanism of protection in experimental pneumococcal bacteremia.</a> J. Immunol. 133, 3308-3312 (1984).</li><li>Paciorkowski, N., Porte, P., Shultz, L. D., Rajan, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B1+B+lymphocytes+play+a+critical+role+in+host+protection+against+lymphatic+filarial+parasites.">B1 B lymphocytes play a critical role in host protection against lymphatic filarial parasites.</a> J. Exp. Med. 191, 731-736 (2000).</li><li>Mercolino, T. J., Arnold, L. W., Hawkins, L. A., Haughton, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+mouse+peritoneum+contains+a+large+population+of+Ly-1++(CD5)+B+cells+that+recognize+phosphatidyl+choline:+relationship+to+cells+that+secrete+hemolytic+antibody+specific+for+autologous+erythrocytes.">Normal mouse peritoneum contains a large population of Ly-1+ (CD5) B cells that recognize phosphatidyl choline: relationship to cells that secrete hemolytic antibody specific for autologous erythrocytes.</a> J. Exp. Med. 168, 687-698 (1988).</li><li>O'Garra, A., Chang, R., Go, N., Hastings, R., Haughton, G., Howard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ly-1+B+(B-1)+cells+are+the+main+source+of+B+cell+derived+interleukin+10.">Ly-1 B (B-1) cells are the main source of B cell derived interleukin 10.</a> Eur. J. Immunol. 22, 711-717 (1992).</li><li>Hoover, D. L., Nacy, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+activation+to+kill+Leishmania+tropica:+Defective+intracellular+killing+of+amastigotes+by+macrophages+elicited+with+sterile+inflammatory+agents.">Macrophage activation to kill Leishmania tropica: Defective intracellular killing of amastigotes by macrophages elicited with sterile inflammatory agents.</a> J. Immunol. 132, 1487-1491 (1984).</li><li>Austin, P. E., McCulloch, E. A., Till, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+factor+in+L-cell+conditioned+medium+capable+of+stimulating+colony+formation+by+mouse+marrow+cells+in+culture.">Characterization of the factor in L-cell conditioned medium capable of stimulating colony formation by mouse marrow cells in culture.</a> J. Cell Physiol. 77, 121-134 (1971).</li><li>Stanley, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+macrophage+colony-stimulating+factor,+CSF-1.">The macrophage colony-stimulating factor, CSF-1.</a> Methods Enzymol. 116, 564-587 (1985).</li><li>Baumgarth, N., Chen, J., Herman, O. C., Jager, G. C., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+B-1+and+B-2+cells+in+immune+protection+from+influenza+virus+infection.">The role of B-1 and B-2 cells in immune protection from influenza virus infection.</a> Curr. Top. Microbiol. Immunol. 252, 163-169 (2000).</li><li>Baumgarth, N., Herman, O. C., Jager, G. C., Brown, L. E., Herzenberg, L. A., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-1+and+B-2+cell-derived+immunoglobulin+M+antibodies+are+nonredundant+components+of+the+protective+response+to+influenza+virus+infection.">B-1 and B-2 cell-derived immunoglobulin M antibodies are nonredundant components of the protective response to influenza virus infection.</a> J. Exp. Med. 192, 271-280 (2000).</li><li>Berland, R., Wortis, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+and+functions+of+B-1+cells+with+notes+on+the+role+of+CD5.">Origins and functions of B-1 cells with notes on the role of CD5.</a> Annu. Rev. Immunol. 20, 253-300 (2002).</li></ol>

腹腔细胞的分离，不同的免疫细胞，主要是巨噬细胞和特定的B细胞亚群的研究是一个重要的技术。虽然这是一个简单的过程，也有一些涉及的关键步骤。应避免污染所收集的样品中血液的存在，获得纯腹腔细胞群。颈椎脱位安乐死应做仔细，以避免在腹腔的血液污染。或者可以使用的CO 2。此外，在手术过程中适当的照顾中应采取，以避免在腹腔穿刺膀胱或任何其他机关。注入流体恢复大部分也是很重要的细胞产量。 此过程被广泛用于研究自中等数量的居民巨噬细胞生物学，未刺激巨噬细胞可以很容易地获得从腹腔，在髓系祖细胞在体外分化成成熟的巨噬细胞，巨噬细胞集落刺激因子（8费力的任务，9）。从腹腔巨噬细胞的产量可以提高使用硫乙醇酸盐启发式，虽然硫乙醇酸盐的使用可能会改变的巨噬细胞的生理特性（7）。 B细胞是先天和适应性免疫中扮演关键角色，我们的免疫系统的重要组成部分。在不同的B细胞亚群，B1细胞构成独特的先天免疫，自身免疫和免疫调节B细胞参与的一个子集。 B1细胞主要位于腹腔和自我补充。它们是自然的IgM它提供了针对病毒和细菌（10-12）保护的第一线的主要生产商之一。 B1细胞的IL - 10（6），这是一个重要的细胞因子参与免疫调节的主要来源。虽然几项研究已经与B1单元格的追求，仍然有余地为进一步评价其​​对比功能，特别是其监管作用。腹腔细胞的分离方法研究B1细胞的功能提供了独特的机会。

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isolation of mouse peritoneal cavity cells

腹膜腔是腹腔膜结合的充满液体的哺乳动物，其中包含的肝，脾，大部分的胃肠道和其他脏器。港口包括巨噬细胞，B细胞和T细胞的免疫细胞的数量。一个天真腹腔巨噬细胞大量存在，使一个天真的组织驻地巨噬细胞（1）收集的首选网站。腹膜腔是因为存在一个独特的腹腔居民的B细胞为B1细胞除了常规B2细胞的一个子集B细胞的研究也很重要。 B1细胞分为菌素和B1B细胞，它可以通过表面的CD11b和CD5表达尊敬。 B1单元格是一个由多种病原体（2-4）早期保护自然提供的IgM的重要来源。这些细胞是自身反应（5）性质，但它们是如何控制，以防止自身免疫性疾病仍然是不完全理解。相反，CD5<sup&gt; +</sup凭借他们的IL - 10的生产能力（6）&gt;阿维菌素B1a的细胞具有一定的调节特性。因此，腹腔B1细胞研究，因为他们的不同的功能和与他们的发展及规管相关的许多未解决的问题是一个有趣的细胞群。腹腔居民的免疫细胞的分离是非常棘手，因为缺乏一个定义的结构腹腔内。我们的协议，将描述一个获得可行的免疫细胞，从小鼠的腹腔，然后可以使用的表型分析，流式细胞仪和不同的生化和免疫学检测程序。

Watch this Scientific Journal Video about Isolation of Mouse Peritoneal Cavity Cells at JoVE.com

Isolation of Mouse Peritoneal Cavity Cells

在哺乳动物的腹腔中包含不同的免疫细胞群的先天免疫反应的关键。这些细胞的生化和功能分析的一个有效的隔离方法是必需的。在这里，我们提供一个全面的方法在小鼠腹腔细胞隔离。

小鼠腹膜腔细胞的分离

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

isolation-of-mouse-peritoneal-cavity-cells

Research

JoVE Journal

Immunology and Infection

142.9K 次观看.  Blood Research Institute. 在哺乳动物的腹腔中包含不同的免疫细胞群的先天免疫反应的关键。这些细胞的生化和功能分析的一个有效的隔离方法是必需的。在这里，我们提供一个全面的方法在小鼠腹腔细胞隔离。

视频: Isolation of Mouse Peritoneal Cavity Cells

Isolation of Functional Cardiac Immune Cells

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 include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

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

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 respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

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 Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

小鼠生殖道黏膜淋巴细胞分离

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

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 ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

小鼠腹腔巨噬细胞的分离，以贯彻基因表达分析后Toll样受体刺激

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

从大鼠睾丸支持细胞的分离和管周细胞

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

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 ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

增强型绿色荧光蛋白表达大肠杆菌评价小鼠腹腔巨噬细胞吞噬细胞增多

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. They are part of a family of chemosensory epithelial cells which include tuft cells in the intestinal mucosa, brush cells in the trachea, and solitary chemosensory and microvillous cells in the nasal mucosa. Chemosensory cells in different epithelial compartments share key intracellular markers and a core transcriptional signature, but also display significant transcriptional heterogeneity, likely reflective of the local tissue environment. Isolation of tracheal brush cells from single cell suspensions is required to define the function of these rare epithelial cells in detail, but their isolation is challenging, potentially due to the close interaction between tracheal brush cells and nerve endings or due to airway-specific composition of tight and adherens junctions. Here, we describe a procedure for isolation of brush cells from mouse tracheal epithelium. The method is based on an initial separation of tracheal epithelium from the submucosa, allowing for a subsequent shorter incubation of the epithelial sheet with papain. This procedure offers a rapid and convenient solution for flow cytometric sorting and functional analysis of viable tracheal brush cells.

Brush cells are rare cholinergic chemosensory epithelial cells found in the na&#239;ve mouse trachea. Due to their limited numbers, ex vivo evaluation of their functional role in airway immunity and remodeling is challenging. We describe a method for isolation of tracheal brush cells by flow cytometry.

小鼠特拉切斯刷细胞的分离与定量评价

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. ...