이 작품은 NIH 부여 AI069358 및 BloodCenter 연구 재단에 의해 부분적으로 지원되었다.

<ol><li> 프로세스를 시작하기 전에 다음 항목 수집 및 준비에 필요 : <ul><li> 얼음 </li><li> 27g 바늘로 5ml 주사기 </li><li> 25g 바늘로 5ml 주사기 </li><li> 스티로폼 블록 및 마우스를 장착을위한 핀 </li><li> 장착 블록 배치 수있는 트레이 </li><li> 가위와 집게 </li><li> 컬렉션 튜브 </li><li> 70 % 에탄올 </li><li> PBS로 3퍼센트 소태아 혈청 (FCS) (예약 얼음에 냉장 및 보관) </li></ul></li><li> 마우스를 안락사, 70 % 에탄올로 스프레이하고 뒷면에​​있는 스티로폼 블록에 탑재합니다. </li><li> 가위와 집게를 사용하면 복막의 바깥쪽 피부를 잘라 부드럽게 복막 구멍을 내벽 안쪽 피부를 노출 다시 가져옵니다. </li><li> 27g 바늘을 사용하여 복막 구멍에 얼음 차가운 PBS (3 % FCS 포함) 5 ML을 주사. 어떤 장기 조심하지 펑크로되는 복막에 천천히 바늘을 밀어. </li><li> 주입 후, 부드럽게 PBS 용액에 어떤 부착된 세포를 이동시키다하는 복막 마사지. </li><li> 복막에 5 ML의 주사기에 부착된, 25g 바늘, 베벨을 삽입하고 지방 조직이나 다른 기관에 의해 막히는 것을 방지할 수 부드럽게 바늘의 팁을 이동하면서 유체를 수집합니다. 가능한 한 많은 유체로 수집하고 주사기에서 바늘을 제거한 후에 얼음에 보관 튜브에 수집한 세포 현탁액을 입금. 옵션 : 4-6 단계를 반복 </li><li> 복막의 내부 피부에 절개를 만들고 포셉로 피부를 누른 상태 것은 구멍에서 남아있는 유체를 수집하는 플라스틱 파스퇴르 피펫을 사용합니다. </li><li> 단계에서 6, 7 볼 혈액 오염이 감지되면 다음 오염된 샘플을 폐기해야합니다. </li><li> 8 분 동안 1,500 RPM에서 수집된 세포 현탁액을 스핀 뜨는를 버리고 계산을 위해 원하는 미디어 또는 PBS에 세포를 resuspend. </li></ol> thioglycollat​​e를 얻는 다른 프로토콜 macrophages을 elicited : 이 방법은 macrophages 높은 수율을 얻을하는 데 사용됩니다. <ol><li> 3퍼센트 (W / V) 각 마우스의 복막 구멍에 브루어 thioglycollat​​e 미디어 (7) 5 ML을 주사. </li><li> 3~5일 기다려, 그리고 세포의 수집에 대한 위의 1 단계를 진행합니다. </li></ol> nonelicited 접근 방식에 비해 약 10 배 이상 macrophages가 수집하실 수 있습니다. 유일한 관심사는이 절차에 의해 얻은 macrophages 자신의 생리 특성의 일부가 다르다는 것입니다. 대표 결과 : unmanipulated 마우스, 5-10000000 복막 캐비티 세포는 좋은 격리 프로토콜로 구할 수 있습니다. 모든 생활 세포 가운데 50-60%는 B 세포 있으며, ~ 30 % macrophages 및 50~10%는 T 세포 (그림 1)입니다. <img src="/files/ftp_upload/1488/1488fig1.jpg" alt="그림 1" /> 그림 1. 복막 캐비티으로부터 격리 세포의 표현형. 복막 캐비티 세포들은의 다음과 같은 분리는 물들일되었습니다 방지 마우스 TCRβ - FITC, B220 - PE - 텍사스 빨강, CD11b 태평양 파랑, CD23 - PE - Cy7 및 CD5 - APC. 대표 흐름 cytometric 플롯은 B와 T 세포 (A), macrophages (B), B1과 B2 셀 (C)와 B1a 및 B1b 세포 (D)의 비율을 보여줍니다.

<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>

복막 캐비티 세포의 격리는 다른 면역 세포, 주로 macrophages 및 구체적인 B 세포 하위 집합의 연구를위한 중요한 기술이다. 이 간단한 과정이지만, 관련된 몇 가지 중요한 단계가 있습니다. 수집된 샘플의 혈액을 오염의 존재는 순수 복막 캐비티 세포 인구를 얻을 피해야한다. 자궁 경부 전위에 의해 안락사는 복막에 고인 혈액 오염을 피하기 위해 신중하게 이루어져야합니다. 또는 CO 2를 사용할 수 있습니다. 또한, 프로 시저 적절한 치료 도중 복막 구멍에있는 방광이나 다른 장기 펑쳐링 않도록 주의해야한다. 주입 액의 대부분을 복구하면 세포 수율하는 것이 중요합니다. 이 절차는 널리 주민의 중간 번호부터 대식 세포 생물학을 연구하는 데 사용됩니다, unstimulated macrophages 쉽게 대식 세포 콜로니 자극 인자 (8을 사용하여 체외에서 성숙 macrophages에 골수양 전구 세포를 차별 힘드는 작업과는 달리, 복막 캐비티에서 얻을 수 있습니다 , 9). thioglycollat​​e 사용이 macrophages (7)의 생리적 특성을 변경할 수도 있지만 복막 캐비티에서 대식 세포 수율은 thioglycollat​​e의 이끌어 냄 메서드를 사용하여 향상시킬 수 있습니다. B 세포는 타고난과 적응 면역 모두에 중요한 역할을 우리 면역 체계의 중요한 부분입니다. 다른 B 세포 subpopulations 중에서 B1 세포는 타고난 면역, autoimmunity 및 면역 조절에 관여 B 세포의 고유한 하위 집합을 구성. B1 세포는 주로 복막 캐비티에 위치하고 있으며 자체 replenishing입니다. 그들은 바이러스의 번호와 박테리아 (10-12)에 대한 보호의 첫 번째 라인을 제공하고 자연 IgM의 주요 생산자 중 하나이다. B1 세포는 또한 면역 조절에 관여하는 중요한 시토킨는 IL - 10 (6)의 주요 원천입니다. 여러 연구가 B1 세포와 함께 추진되고 있지만, 아직 특별히 자신의 대조 기능을 추가로 평가를 위해 규제의 역할 범위가있다. 복막 캐비티 세포 분리 방법은 B1 세포의 기능을 연구하는 독특한 기회를 제공합니다.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Thioglycollate Medium, Brewer Modified</td>
<td>&nbsp;</td>
<td>BD BBL</td>
<td>211716</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation of mouse peritoneal cavity cells

복막 구멍은 간, 비장, gastro - 창자 및 기타 내장의 대부분을 포함하고 포유류의 막 - 바운드와 유체 가득한 복강입니다. 그것은 macrophages, B 세포와 T 세포를 포함한 면역 세포의 숫자를 항구. 복막 캐비티에 순진한 macrophages 높은 숫자의 존재는 순진 조직 주민 macrophages (1)의 수집을 위해 선호하는 사이트를 만듭니다. 복막 캐비티도 있기 때문에 기존의 B2 셀에 이외에 지하 1 층 세포로 알려진 독특한 복막 캐비티 상주 B 세포 부분 집합의 존재의 B 세포의 연구에 중요합니다. B1 세포는 CD11b와 CD5의 표면 표현으로 구분할 수 B1a하고 B1b 세포로 세분화됩니다. B1 세포가 병원균 (2-4)의 다양한 초기 보호를 제공하는 자연 IgM의 중요한 소스입니다. 이러한 세포는 자연 (5)에 autoreactive 있지만, 그들이 autoimmunity을 방지하기 위해 관리하는 방법은 아직 완전히 이해되지 않습니다. 반대로, CD5에<sup&gt; +</sup&gt; B1a 세포는 IL - 10 생산 용량 (6)을 가진 일부 규제 속성을 보유하고 있습니다. 따라서 복막 공동 B1 세포 때문에 그들의 다양한 기능과 개발 및 규제와 관련된 여러 unaddressed 질문 연구에 흥미로운 세포 인구입니다. 복막 캐비티 주민 면역 세포의 분리 때문에 복막 캐비티 내부에 정의된 구조의 부족 까다롭습니다. 우리 프로토콜은 다음 유동세포계측법에 의해 및 다른 생화 학적 및 면역 assays에 대한 phenotypic 분석을 위해 사용할 수있는 생쥐의 복막 캐비티에서 현실적인 면역 세포를 얻기위한 절차를 설명합니다.

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

Isolation of Mouse Peritoneal Cavity Cells

포유류의 복막 캐비티는 타고난 면역 반응에 중요한 다른 면역 세포 인구가 포함되어 있습니다. 효율적인 분리 방법은 이러한 세포의 생화 학적 및 기능적 분석이 필요합니다. 여기서 우리는 마우스의 복막 공동 세포의 격리를위한 포괄적인 방법을 제공합니다.

마우스 복막 구멍 세포의 분리

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

142.8K 조회수.  Blood Research Institute. 포유류의 복막 캐비티는 타고난 면역 반응에 중요한 다른 면역 세포 인구가 포함되어 있습니다. 효율적인 분리 방법은 이러한 세포의 생화 학적 및 기능적 분석이 필요합니다. 여기서 우리는 마우스의 복막 공동 세포의 격리를위한 포괄적인 방법을 제공합니다.

비디오: 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.

심장 기능 면역 세포의 분리

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 ...

연구

면역학 및 감염병학

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.

마우스 폐에 돌기 세포의 분리

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.

마우스 소장에서 돌기 세포 및 Macrophages의 분리 및 특성화

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

마우스 성기 요로 점막에서 림프구의 분리

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.

지방 조직 면역 세포의 분리

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.

쥐 복막 대 식세포의 분리는 수신자 같은 수용체 자극하면 유전자 발현 분석을 수행하기

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.

쥐 고환의 세르 톨리 (Sertoli) 세포의 분리 및 세뇨관 세포

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.

허혈성 마우스 뇌에서 면역 세포의 분리 및 유세포 분석

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.

녹색 형광 단백질 표현 하는 향상 된 사용 하 여 대장균 마우스 복 막 대 식 세포 식 균 작용을 평가 하기 위해

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.

마우스 기관에서 브러시 셀의 격리 및 정량 평가

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

Methods Article

마우스 복막 구멍 세포의 분리