Ce travail a été soutenu en partie par NIH AI069358 et la Fondation de recherche BloodCenter.

<ol><li> Avant de commencer le processus, les éléments suivants doivent être recueillis et préparés à: <ul><li> Glace </li><li> Seringue de 5 ml avec aiguille 27G </li><li> Seringue de 5 ml avec aiguille 25g </li><li> Styrofoam blocs et des épingles pour le montage de la souris </li><li> Plateau sur lequel le bloc de montage peut être placé </li><li> Ciseaux et pinces </li><li> Tubes de prélèvement </li><li> 70% d&#39;éthanol </li><li> PBS avec 3% sérum de veau foetal (FCS) (pré refroidis et conservés sur glace) </li></ul></li><li> Euthanasier la souris, le vaporiser avec de l&#39;éthanol 70% et le monter sur le bloc de styromousse sur le dos. </li><li> En utilisant une paire de ciseaux et des pinces à couper la peau externe du péritoine et tirez en arrière pour exposer la peau intérieure tapissant la cavité péritonéale. </li><li> Injecter 5 ml de PBS glacé (avec 3% de FCS) dans la cavité péritonéale à l&#39;aide d&#39;une aiguille 27G. Poussez l&#39;aiguille lentement dans le péritoine en faisant attention à ne pas percer tous les organes. </li><li> Après l&#39;injection, masser doucement le péritoine pour déloger les cellules attachées dans la solution PBS. </li><li> Insérer une aiguille de 25 g, biseau vers le haut, attachée à une seringue de 5 ml dans le péritoine et recueillir le liquide tout en déplaçant la pointe de l&#39;aiguille doucement pour éviter le colmatage par le tissu adipeux ou d&#39;autres organes. Recueillir autant de liquide que possible et le dépôt de la suspension cellulaire recueilli dans des tubes conservés sur la glace après avoir retiré l&#39;aiguille de la seringue. En option: 4-6 Répétez l&#39;étape </li><li> Faire une incision dans la peau intérieure du péritoine et tout en maintenant la peau avec une pince utiliser une pipette Pasteur en plastique pour recueillir le liquide restant dans la cavité. </li><li> Si à l&#39;étape 6 ou 7 contamination du sang visible est détectée, alors l&#39;échantillon contaminé doit être jeté. </li><li> Faites tourner la suspension de cellules recueillies à 1500 RPM pendant 8 minutes, éliminer le surnageant et remettre en suspension les cellules dans les médias souhaités ou PBS pour le comptage. </li></ol> Protocole alternatif pour obtenir thioglycolate suscité des macrophages: Cette méthode est utilisée pour obtenir un rendement plus élevé des macrophages. <ol><li> Injecter 5 ml de 3% (p / v) Brewer milieu au thioglycolate (7) dans la cavité péritonéale de chaque souris. </li><li> Attendre pendant 3-5 jours, et passez à l&#39;étape 1 ci-dessus pour la collecte des cellules. </li></ol> Comparativement à l&#39;approche nonelicited, environ 10 fois plus de macrophages peuvent être collectées. Le seul souci est que les macrophages obtenus par cette procédure diffèrent dans certaines de leurs propriétés physiologiques. Les résultats représentatifs: De une souris non manipulée, 5-10 millions de cellules cavité péritonéale peut être obtenue avec un protocole de bonne isolation. Parmi toutes les cellules vivantes, 50-60% sont des cellules B, macrophages ~ 30% et 5-10% sont des cellules T (figure 1). <img src="/files/ftp_upload/1488/1488fig1.jpg" alt="Figure 1" /> Figure 1. Phénotype des cellules isolées de la cavité péritonéale. Isolement de cellules Après la cavité péritonéale, ils ont été colorés avec anti-souris-FITC TCRβ, B220-PE-rouge Texas, CD11b-Pacifique bleu, CD23-PE-Cy7 et CD5-APC. Parcelles de flux représentant cytométrie montrent des pourcentages de cellules B et T (a), les macrophages (b), B1 et B2 cellules (c) et les cellules B1a et B1b (d).

<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>

Isolement de cellules cavité péritonéale est une technique importante pour l&#39;étude des différentes cellules du système immunitaire, principalement les macrophages et les sous-ensembles spécifiques de cellules B. Bien que ce processus est simple, il ya quelques étapes essentielles impliquées. Présence de contamination du sang dans l&#39;échantillon prélevé doit être évitée afin d&#39;obtenir un pur population cellulaire péritonéale cavité. L&#39;euthanasie par dislocation cervicale doit être effectuée avec soin pour éviter toute contamination du sang dans la cavité péritonéale. Alternativement CO 2 peuvent être utilisés. En outre, pendant les soins procédure appropriée devrait être prise pour éviter la perforation de la vessie ou d&#39;autres organes dans la cavité péritonéale. Récupérer la plupart du liquide injecté est également important pour le rendement cellulaire. Cette procédure est largement utilisée pour étudier la biologie des macrophages, depuis un nombre modéré de résident, les macrophages non stimulés peuvent facilement être obtenus à partir de la cavité péritonéale, contrairement à la tâche laborieuse de différenciation des cellules progénitrices myéloïdes en macrophages matures in vitro, en utilisant le facteur de croissance macrophages (8 , 9). Le rendement des macrophages de la cavité péritonéale peut être améliorée en utilisant la méthode de l&#39;élicitation thioglycolate, bien que l&#39;utilisation thioglycolate pourraient altérer les propriétés physiologiques des macrophages (7). Les cellules B sont une partie importante de notre système immunitaire joue un rôle crucial dans les deux immunité innée et adaptative. Parmi les différentes sous-populations de cellules B, les cellules B1 comprend un sous-ensemble unique de cellules B impliquées dans l&#39;immunité innée, l&#39;auto-immunité et de la régulation immunitaire. Cellules B1 sont principalement situés dans la cavité péritonéale et sont la reconstitution de soi. Ils sont l&#39;un des principaux producteurs d&#39;IgM naturel qui assure la première ligne de protection contre un certain nombre de virus et de bactéries (10-12). Cellules B1 sont aussi une source importante d&#39;IL-10 (6), qui est une cytokine importante impliquée dans la régulation immunitaire. Bien que plusieurs études ont été menées avec les cellules B1, encore il ya place pour une évaluation plus poussée de leurs fonctions contrastées, spécialement leur rôle de régulation. Péritonéale méthode d&#39;isolement cavité cellulaire offre l&#39;opportunité unique d&#39;étudier les fonctions des cellules B1.

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<td>Thioglycollate Medium, Brewer Modified</td>
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isolation of mouse peritoneal cavity cells

La cavité péritonéale est une cavité membranaires et remplis de fluide abdominal de mammifères, qui contient le foie, la rate, la plupart du tractus gastro-intestinal et d&#39;autres viscères. Il abrite un certain nombre de cellules immunitaires, y compris les macrophages, les lymphocytes B et les lymphocytes T. La présence d&#39;un nombre élevé de macrophages naïfs dans la cavité péritonéale en fait un site privilégié pour la collecte des naïfs macrophages résidents du tissu (1). La cavité péritonéale est également important pour l&#39;étude des cellules B en raison de la présence d&#39;une cavité péritonéale unique de sous-résident de cellules B ou cellules B1, en plus de cellules B2 conventionnels. Cellules B1 sont subdivisées en cellules B1a et B1b, qui peut être distingué par l&#39;expression de surface de CD11b et CD5. Cellules B1 sont une source importante d&#39;IgM naturels offrant une protection précoce d&#39;une variété de pathogènes (2-4). Ces cellules sont autoréactifs dans la nature (5), mais comment ils sont contrôlés pour éviter l&#39;auto-immunité n&#39;est pas encore parfaitement compris. Au contraire, CD5<sup&gt; +</sup&gt; Cellules B1a possèdent certaines propriétés de régulation en vertu de leur capacité de l&#39;IL-10 produit (6). Par conséquent, les cellules B1 péritonéale cavité sont une population de cellules intéressantes à étudier en raison de leurs fonctions diverses et nombreuses questions sans réponse associés à leur développement et de régulation. L&#39;isolement de la cavité péritonéale des cellules immunitaires résidentes est délicat en raison du manque d&#39;une structure définie à l&#39;intérieur de la cavité péritonéale. Notre protocole se décrivent une procédure pour l&#39;obtention de cellules viables à l&#39;abri de la cavité péritonéale de souris, qui peuvent ensuite être utilisés pour l&#39;analyse phénotypique par cytométrie en flux et pour différents dosages biochimiques et immunologiques.

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

Isolation of Mouse Peritoneal Cavity Cells

La cavité péritonéale chez les mammifères contient différentes populations de cellules immunitaires crucial pour les réponses immunitaires innées. Une méthode d&#39;isolation efficace est nécessaire pour les analyses biochimiques et fonctionnelles de ces cellules. Ici nous fournissons une méthode globale pour l&#39;isolement des cellules cavité péritonéale chez la souris.

L&#39;isolement des cellules de souris cavité péritonéale

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

142.6K vues.  Blood Research Institute. La cavité péritonéale chez les mammifères contient différentes populations de cellules immunitaires crucial pour les réponses immunitaires innées. Une méthode d'isolation efficace est nécessaire pour les analyses biochimiques et fonctionnelles de ces cellules. Ici nous fournissons une méthode globale pour l'isolement des cellules cavité péritonéale chez la souris.

Vidéo: 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.

Isolement des cellules immunitaires fonctionnelles cardiaques

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 ...

Recherche

Immunologie et infection

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.

L&#39;isolement des cellules de souris poumon dendritiques

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.

L&#39;isolement et la caractérisation des cellules dendritiques et les macrophages de l&#39;intestin de souris

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

Isolement des lymphocytes de souris muqueuses génitales Tract

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.

Isolement de tissu adipeux cellules immunitaires

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.

Isolement d&#39;anticorps murins péritonéale macrophages pour effectuer Analyse expression génique Sur Toll-like récepteurs Stimulation

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.

Isolement des cellules de Sertoli et péritubulaire cellules de rat Testicules

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.

Isolement et analyse par cytométrie en flux des cellules immunitaires du cerveau ischémique souris

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.

Utilisant amélioré Fluorescence verte exprimant protéines Escherichia Coli pour évaluer la phagocytose des macrophages péritonéaux de souris

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.

Isolement et évaluation quantitative des cellules de brosse de La souris trachée

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