この作品は、NIHの助成金AI069358とBloodCenter研究財団によって部分的にサポートされていました。

<ol><li>プロセスを開始する前に、以下の項目は、収集と準備に注意する必要があります。 <ul><li>アイス</li><li> 27グラムの針で5ミリリットル注射器</li><li> 25グラムの針で5ミリリットル注射器</li><li>発泡スチロールブロックとマウスを取り付けるためのピン</li><li>取り付けブロックを置くことができる上トレイ</li><li>はさみや鉗子</li><li>コレクションチューブ</li><li> 70％エタノール</li><li> PBSで3％ウシ胎児血清（FCS）（前は氷上で冷却して保持） </li></ul></li><li>マウスを安楽死させる、70％エタノールでそれをスプレーし、その背面に発泡スチロールのブロックにマウントします。 </li><li>ハサミとピンセットを使用すると、腹膜の外側の皮膚をカットし、静かに腹腔の内側を覆う内側の肌を露出するために、それを引き戻す。 </li><li> 27グラム針を用いて腹腔内に、氷冷PBS（3％FCSを含む）5 mlを注入する。ない穿刺あらゆる臓器にまで注意して腹膜にゆっくりと針を押し込みます。 </li><li>注入後、軽くPBS溶液に接続されているすべての細胞を取り除くために、腹膜をマッサージ。 </li><li>腹膜の5 mlシリンジに接続されて、25gの針、ベベルを挿入し、脂肪組織や他の臓器で目詰まりを避けるために、静かに針の先端を移動させながら液体を集める。できる限り多くの流体を収集し、シリンジから針を除去した後、氷上に置きチューブに回収細胞懸濁液を入金。 オプション：ステップ4-6を繰り返します。 </li><li>腹膜の内側の皮膚の切開を行うと鉗子で皮膚を押しながらすると空洞から残りの液を収集するためにプラスチック製パスツールピペットを使用してください。 </li><li>ステップで6または7目に見える血液汚染が検出された場合、汚染されたサンプルは破棄されるべきである。 </li><li> 、8分間1500rpmで採取した細胞懸濁液をスピン上清を破棄し、カウントするために必要なメディアまたはPBSで細胞を懸濁します。 </li></ol>チオグリコール酸を得るために代替プロトコルは、マクロファージを誘発した。 このメソッドは、マクロファージのより高い収量を得るために使用されます。 <ol><li> 3％（w / v）の各マウスの腹腔内にブリューチオグリコール酸培地（7）5 mlを注入する。 </li><li> 3-5日を待って、セルのコレクションのために上記のステップ1へ進みます。 </li></ol> nonelicitedアプローチに比べ、約10倍以上のマクロファージを採取することができます。唯一の懸念は、この手順で得られたマクロファージはそれらの生理学的特性のいくつかで異なることです。 代表的な結果： unmanipulatedマウスから、5〜10000000腹腔細胞は、良好なアイソレーションプロトコルで取得できます。すべての生細胞の中で、50〜60％がB細胞である、〜30％のマクロファージと5〜10％は、T細胞（図1）です。 <img src="/files/ftp_upload/1488/1488fig1.jpg" alt="図1" /> 図1。腹膜腔から単離した細胞の表現型。それらは抗マウスTCRβ- FITC、B220 - PE -テキサスレッド、CD11bパシフィックブルー、CD23 - PE - Cy7とCD5 - APCで染色した腹腔細胞の以下の単離。代表的なフローサイトメトリープロットは、BおよびT細胞（）、マクロファージ（B）、B1とB2セル（C）とB1AとB1B細胞（D）の割合を示しています。

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

腹腔細胞の分離は、異なる免疫細胞、主としてマクロファージおよび特定のB細胞サブセットの研究のための重要なテクニックです。これは単純なプロセスですが、関係するいくつかの重要なステップがあります。採取したサンプル中の血液を汚染の存在は純粋な腹腔内の細胞集団を得るために避けるべきである。頚椎脱臼による安楽死は、腹腔内の血液の汚染を避けるために慎重に行う必要があります。あるいはCO 2を使用することができます。さらに、手順の間に適切なケアは、膀胱や腹腔内の他の臓器を穿刺しないように注意してください。注入された流体のほとんどを回復すると、セルの歩留まりに重要です。 この手順は、広く住民の中程度の数字からマクロファージ生物学を研究するために使用され、刺激されていないマクロファージを簡単にマクロファージコロニー刺激因子（8を使用して、in vitroで成熟したマクロファージに骨髄性前駆細胞を分化させる骨の折れる作業とは対照的に、腹腔から得ることができます。 、9）。チオグリコール酸の使用は、マクロファージ（7）の生理学的特性を変えるかもしれませんが、腹腔からマクロファージの収率は、チオグリコール酸誘発法を用いて改善することができます。 B細胞は自然免疫と獲得免疫の両方において重要な役割を担う私たちの免疫システムの重要な部分です。異なるB細胞亜集団の中で、B1細胞は自然免疫、自己免疫と免疫調節に関与するB細胞のユニークなサブセットで構成されています。 B1細胞は主に腹腔内に位置しており、自己補給がありますされています。彼らは、ウイルスと細菌の数に対する保護の最初の行を提供する（10-12）自然IgMの主要生産国の一つです。 B1細胞は、免疫調節に関与する重要なサイトカインであるIL - 10（6）、の主要な源です。いくつかの研究がB1細胞と追求されているが、まだ特別に、その対照的な機能のさらなる評価のために彼らの調節の役割範囲があります。腹腔細胞の単離方法は、B1細胞の機能を研究するユニークな機会を提供します。

<!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

腹腔、肝臓、脾臓、消化管や他の内臓のほとんどが含まれている哺乳類の膜結合型と液体で満たされた腹腔内、です。それは、マクロファージ、B細胞とT細胞を含む免疫細胞の数を庇護する。腹腔内のナイーブなマクロファージの数が多いの存在は、そのナイーブな組織の常駐マクロファージ（1）のコレクションのための好ましいサイトです。腹膜腔には、理由、従来のB2セルに加えて、B1細胞として知られているユニークな腹腔内に常駐するB細胞のサブセットの存在のB細胞の研究に重要である。 B1細胞はCD11bとCD5の表面発現により区別することができますB1AとB1B細胞に分割されています。 B1細胞は病原体（2-4）の様々な早期保護を提供する自然IgMの重要な源です。これらの細胞は、自然（5）の自己反応性であるが、それらが自己免疫を防ぐために制御される方法はまだ完全には解明されていない。逆に、CD5について<sup&gt; +</sup&gt; B1A細胞はそのIL - 10産生能力（6）のおかげで、いくつかの規制性質を有している。したがって、腹腔内のB1セルのは、その多様な機能とそれらの発展と規制に関連する多く取り組まれていない問題の研究に興味深い細胞集団です。腹腔内居住者の免疫細胞の分離が原因腹腔内部に定義された構造の不足の注意が必要です。我々のプロトコルは、フローサイトメトリーにより、異なる生化学的および免疫学的アッセイのための表現型解析に使用できるマウスの腹膜腔、より現実的な免疫細胞を得るための手順を説明します。

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

Isolation of Mouse Peritoneal Cavity Cells

哺乳類の腹腔内には、自然免疫応答のための重要な別の免疫細胞集団が含まれています。効率的な単離法は、これらの細胞の生化学と機能解析のために必要です。ここでは、マウスの腹腔内細胞の単離のための包括的な方法を提供する。

マウス腹腔細胞の分離

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 ビュー.  Blood Research Institute. 哺乳類の腹腔内には、自然免疫応答のための重要な別の免疫細胞集団が含まれています。効率的な単離法は、これらの細胞の生化学と機能解析のために必要です。ここでは、マウスの腹腔内細胞の単離のための包括的な方法を提供する。

ビデオ: 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.

機能心臓の免疫細胞の単離

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 ...

免疫・感染学

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.

マウス肺樹状細胞の分離

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.

マウスの腸からの樹状細胞およびマクロファージの単離とキャラクタリゼーション

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

マウス生殖管粘膜からのリンパ球の単離

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

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

脂肪組織の免疫細胞の単離

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.

Toll様受容体刺激時の遺伝子発現解析を行うためにマウス腹腔マクロファージの単離

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.

ラット精巣からのセルトリ細胞と尿細管周囲細胞の単離

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.

虚血性マウス脳からの免疫細胞の単離およびフローサイトメトリー分析

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.

緑色蛍光タンパク質-表現する拡張を使用するマウス腹腔マクロファージの貪食能を評価するために大腸菌

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.

マウス気管からのブラシ細胞の単離と定量的評価

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