Эта работа была выполнена при частичной поддержке гранта NIH AI069358 и Фонда BloodCenter исследований.

<ol><li> Перед началом процесса, следующие элементы должны быть собраны и подготовлены: <ul><li> Лед </li><li> 5 мл шприц с иглой 27 г </li><li> 5 мл шприц с иглой 25g </li><li> Пенополистирол блока и булавки для крепления мыши </li><li> Лоток, на котором монтажный блок может быть размещен </li><li> Ножницы и пинцеты </li><li> Коллекция труб </li><li> 70% этанола </li><li> PBS с 3% эмбриональной телячьей сыворотки (FCS) (предварительно охлажденных и держится на льду) </li></ul></li><li> Эвтаназии мыши, распылить это с 70% этанола и закрепить его на блок пенополистирола на спине. </li><li> Использование ножницы и щипцы сократить внешнюю оболочку брюшины и осторожно потяните его назад подвергать кожу внутренней подкладкой брюшную полость. </li><li> Inject 5 мл ледяной PBS (с 3% FCS) в брюшную полость использованием 27 г иглы. Нажимаем иглу медленно в брюшину стараясь не прокол любого из органов. </li><li> После инъекции массируют брюшины выбить все подключенные клетки в раствор PBS. </li><li> Вставьте иглу 25 г, конические вверх, прилагается к 5 мл шприца в брюшину и сбора жидкости при перемещении кончика иглы осторожно, чтобы избежать засорения жировой ткани или других органов. Сбор столько жидкости, сколько возможно, и депозит собраны клеточной суспензии в трубках держится на льду после удаления иглы от шприца. Дополнительно: Повторите шаг 4-6 </li><li> Сделайте разрез в коже внутренней брюшины и, удерживая до кожу щипцами использовать пластиковые пипетки Пастера, чтобы собрать остатки жидкости из полости. </li><li> Если в шаге 6 или 7 видимого загрязнения крови обнаруживается то загрязненные образцы должны быть отброшены. </li><li> Спиновые собраны клеточной суспензии при 1500 оборотов в течение 8 минут, удалите супернатант и ресуспендирования клеток в желаемый носитель или PBS для подсчета. </li></ol> Альтернативный протокол для получения тиогликолята вызвало макрофагов: Этот метод используется для получения высокой доходности макрофагов. <ol><li> Inject 5 мл 3% (м / о) Брюер тиогликолята средний (7) в брюшную полость каждого мыши. </li><li> Ждите в течение 3-5 дней, и переходите к шагу 1 выше, для сбора клеток. </li></ol> По сравнению с nonelicited подход, примерно в 10 раз больше макрофаги могут быть собраны. Единственной заботой является то, что макрофаги, полученные этим процедуры отличаются в некоторых из своих физиологических свойств. Представитель результаты: С unmanipulated мыши, 5-10 млн. перитонеальных клеток полости могут быть получены с хорошими протокол изоляции. Среди всех живых клеток, 50-60% составляют В-клетки, ~ 30% макрофагов и 5-10% являются Т-клетки (рис. 1). <img src="/files/ftp_upload/1488/1488fig1.jpg" alt="Рисунок 1" /> Рисунок 1. Фенотип клеток, выделенных из брюшной полости. После изоляции перитонеальных клеток полости они были окрашены антимышиным TCRβ-FITC, B220-PE-Техас красный, CD11b-Тихоокеанского синий, CD23-PE-Cy7 и CD5-APC. Представитель проточной цитометрии графики показывают процент B и Т-клетки (а), макрофагов (б), B1 и B2 клеток (с) и B1a и B1b клеток (г).

<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>

Выделение перитонеальных клеток полости важный метод для изучения различных иммунных клеток, в первую очередь макрофаги и конкретные подмножества клеток. Хотя это простой процесс, Есть некоторые критические этапы. Наличие загрязняющих крови в собранных образцов следует избегать, чтобы получить чистые популяции клеток перитонеальной полости. Эвтаназия цервикальным сдвигом должно быть сделано аккуратно, чтобы избежать загрязнения крови в брюшной полости. Кроме CO 2 может быть использован. Кроме того, во время процедуры надлежащего ухода должны быть приняты, чтобы избежать проколов пузырь или любых других органов в брюшную полость. Восстановление большинство вводимой жидкости также имеет важное значение в ячейку доходности. Эта процедура широко используется для изучения биологии макрофагов с умеренным количеством житель, нестимулированных макрофаги могут быть легко получены из брюшной полости, в отличие от трудоемкой задачей дифференциации миелоидных клеток-предшественников в зрелые макрофаги в пробирке, используя макрофаг колониестимулирующий фактор (8 , 9). Макрофагов выход из брюшной полости может быть улучшено с помощью метода тиогликолята выявление, хотя тиогликолята использование могло бы изменить физиологические свойства макрофагов (7). В-клетки являются важной частью нашей иммунной системы играет решающую роль в обоих врожденного и адаптивного иммунитета. Среди различных субпопуляций В-клеток, В1 клетки составляют уникальное подмножество B клеток, участвующих в врожденного иммунитета, аутоиммунные заболевания и иммунной регуляции. В1 клетки в основном расположены в брюшной полости и не требуют пополнения. Они являются одним из первичных производителей природного IgM, которая обеспечивает первую линию защиты против ряда вирусов и бактерий (10-12). В1 клетки также основным источником IL-10 (6), который является важным цитокинов, участвующих в иммунной регуляции. Хотя некоторые исследования были проводиться с В1 клетки, по-прежнему есть возможности для дальнейшей оценки их контрастные функций, специально их регулирующей роли. Брюшную полость изолятор метод дает уникальную возможность изучить функции B1 клеток.

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isolation of mouse peritoneal cavity cells

Брюшной полости является мембраной и заполненные жидкостью брюшной полости млекопитающих, в котором содержится в печени, селезенке, большинство из желудочно-кишечного тракта и других внутренних органов. Она таит в себе число иммунных клеток, включая макрофаги, В-клетки и Т-клеток. Наличие большого числа наивных макрофагов в брюшную полость делает его предпочтительным местом для сбора наивно ткани резидент макрофагов (1). Брюшной полости также имеет важное значение для изучения В-клетки из-за наличия уникальных брюшную полость-нерезиденты В-клеток подмножество известного как B1 клеток в дополнение к обычной ячейки B2. В1 клетки подразделяются на B1a и B1b клеток, которые можно отличить по поверхности выражение CD11b и CD5. В1 клетки являются важным источником природных IgM обеспечения раннего защиту от разнообразных патогенов (2-4). Эти клетки аутореактивных в природе (5), но как они находятся под контролем, чтобы предотвратить аутоиммунные до сих пор не поняли полностью. Напротив, CD5<sup&gt; +</sup&gt; B1a клетки обладают некоторыми регуляторными свойствами в силу их Ил-10 производственных мощностей (6). Таким образом, клетки перитонеального B1 полости интересные популяции клеток для изучения из-за их различных функций и много нерешенных вопросов, связанных с их развитием и регулированием. Изоляции брюшной полости резидент иммунных клеток является сложным из-за отсутствия определенной структуры внутри брюшной полости. Наш протокол опишу процедуру получения жизнеспособных клеток иммунной из брюшной полости мышей, которые затем могут быть использованы для фенотипического анализа методом проточной цитометрии и для различных биохимических и иммунологических анализов.

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

Isolation of Mouse Peritoneal Cavity Cells

Брюшной полости у млекопитающих содержит различные иммунные клеточные популяции важно для врожденного иммунного ответа. Эффективный метод изоляции требуется для биохимических и функциональных анализов этих клеток. Здесь мы предлагаем комплексный метод для изоляции брюшной полости в клетках мыши.

Выделение мышью перитонеальных клеток полости

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

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Immunology and Infection

142.7K Views.  Blood Research Institute. Брюшной полости у млекопитающих содержит различные иммунные клеточные популяции важно для врожденного иммунного ответа. Эффективный метод изоляции требуется для биохимических и функциональных анализов этих клеток. Здесь мы предлагаем комплексный метод дл...

Video: 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.

Выделение функциональных сердечно иммунных клеток

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 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.

Выделение мышиные клетки легких Дендритные

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.

Выделение и характеристика дендритных клеток и макрофагов из кишечника мыши

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

Выделение лимфоцитов из половых путей мышь Mucosa

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.

Изоляция жировой ткани иммунных клеток

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.

Выделение перитонеальных макрофагов Carry Out Gene Expression анализ По Toll-подобные рецепторы стимуляции

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.

Выделение клеток Сертоли и перитубулярных Клетки крыс семенников

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.

Выделение и проточной цитометрии анализ иммунных клеток из мозга ишемического Mouse

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.

С помощью усиленной зеленый флуоресценции белков выражая Escherichia Coli оценить мыши перитонеальных макрофагов фагоцитоз

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.

Изоляция и количественная оценка клеток кисти от мыши Tracheas

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