著者らは、ギーセンMeinhardtラボでの一次セルトリ細胞の単離を確立する上で非常に有用だったグイド・バーホーベンとルードDeboel、ルーヴェンを、感謝したいと思います。モニカFijak、ギーセンは、図1Aとアドバイスで彼女の助けのために認められています。研究は、国際研究大学院大学JLUギーセン（ドイツ）/モナッシュ大学（メルボルン、オーストラリア）GRK 1871（SB）の助成金BH 93 / 1-1（SB）および資金調達を通じてドイツ学術協会によってサポートされていました。サポートはまた、「MännlicheInfertilitätバイInfektion＆Entzündung」（MIBIE）と呼ばれるプログラム「ランデス-攻勢ツアEn​​twicklung Wissenschaftlich-ökonomischerExzellenz」（LOEWE）内ヘッセン州の助成金から入手しました。

<ol>
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著者らは、開示することは何もありません。

The seminiferous tubules are bounded by a circular layer of peritubular cells and a basal lamina on the luminal side. Sertoli cells are resting on the basal lamina, establish the blood testis barrier through formation of occluding junctions between adjacent cells and provide the structural framework for the organisation of the seminiferous epithelium. Sertoli cells and adjacent spermatogenic cells maintain intimate contacts throughout germ cell development providing physical contact and communication alike. Therefore, wh...

精巣は、すなわち 、アンドロゲンを男性の配偶子と性ホルモンを産生します。臓器は、二つの区画で構成されています。総精巣容積1の約10〜12％に相当する間質コンパートメントでは、ステロイドはライディッヒ細胞内で起こります。筒状の区画は、精巣容積1の60〜80％に相当し、生殖細胞と体細胞の2種類含まれています-細管周囲細胞とセルトリ細胞を。精巣は、それぞれ1-3高い複雑な精細管を含む、250-300小葉に結合組織隔壁によって分割されます。これらの細管は、基底膜、コラーゲンのシート、及び管周囲細胞（ 図1A）の周層によって囲まれています。 胚上皮は、基底膜の腔側に位置しています。セルトリ細胞は、内腔への基底膜からの全胚上皮にまたがる大規模な細長い細胞です。彼らは強くあるのatt基底膜に痛んだし、間質から胚上皮を吸蔵および血液精巣関門を表す基底外側組織し、タイトジャンクショ​​ンを介して連続セルラーシートを形成。セルトリ細胞は、顕著な細胞質突起及び生殖細胞の種特異的であるが、一定数との緊密な形態学的および機能的な接触に入るためにそれらを有効に波及効果を持っています。二倍体胚性幹細胞は増殖し、精原細胞に分化します。 減数分裂の間に私短命の四倍体精母細胞が減数分裂IIの間に4半数体精子細胞へのさらなる発展が生成されます。それらは細胞のネットを形成するように、すべての生殖細胞質ブリッジによって相互接続されています。精子細胞の成熟の間の主なイベントは、精子と呼ばれるプロセスにおいて、残留体を形成し、細胞質の大部分の押出成形です。残留体はセルトリ細胞によって貪食されます。後期精子細胞は、その後に放出されます管状の内腔とさらに成熟のために精巣上体へ輸送。セルトリ細胞と生殖細胞が相互に地形的および機能的に精子形成を調整するように見えます。 小さ ​​な精巣片を培養し、細胞型顕微鏡2により同定したとき、個々の精巣の細胞型の調製は、ほぼ一世紀前に始まりました。先の細いピンセットを用いて白膜を開いた後の尿細管を注意深く解剖することにより、筒状の間質コンパートメント3を分離するために、後に可能でした。 1975年にウェールズとウィーブは、間質組織と外管周囲細胞層4を除去するためのパンクレアチン処理を付着するの細管を解放するためにコラゲナーゼ処理を導入しました。早期から（古い20日前後）若い未熟ラットの精子形成がまだ始まっていないので、セルトリ細胞は、管状細胞集団の大部分を含んでいる使用されていました。この年齢ラットセルトでOLIの細胞は分裂をやめ、および隣接セル間のタイトジャンクション血液精巣関門が5確立されるように形成します。 ウェールズとウィーブDorringtonのらの独立は、トリプシンの組み合わせを導入し、同年6に出版されたsoybeenトリプシン阻害剤及びコラゲナーゼ処理が続くデオキシ。両グループは、また、約70％のセルトリ細胞を含むめっき均一な細胞懸濁液を生成するために、（それぞれ、シリンジ針またはパスツールピペットを介して繰り返し消化管状フラグメントを描く）機械的な力を用います。培養3日後に、血清を含まない培地を使用して、セルトリ細胞の割合は約90％に増加します。これは主に、生殖細胞を汚染の死に起因する可能性があります。残留管周囲細胞（のPTC）が、しかし、しっかりとそれらの細胞外マトリックスを介して結合したまま。 PTCではなく、セルトリ細胞を産生することが知られていますPTCを用いて汚染を推定するためのマーカータンパク質として働くことができるフィブロネクチン。従って、タンら。追加のヒアルロニダーゼ処理は、セルトリ細胞画分7の純度を向上させるかもしれないと推論しました。実際、それらは、さらなる処置を比較して、血清含有培地を精製したセルトリ細胞を培養するために使用される場合に特に明白となる、約20倍のPTCによる汚染を減少させることができたことを示す可能性があります。その後から桐らの改良された手順に。支配的なプロトコルとなり、広範囲のフィールド8（ 図1B）内の他の主要なグループで使用されていました。 コラゲナーゼ処理中のPTCの大部分が放出され、セルトリ細胞と平行に単離することができます。 PTCを激しく増殖し、卵胞刺激ホルモン（FSH）に応答しないのに対し、セルトリ細胞は、もはや有糸分裂を受け、特徴的な形態学的変化によってFSHに反応しませんおよびサイクリックアデノシン一リン酸（cAMP）濃度9の増加。非常に類似した酵素消化プロトコルは人10、マウス11、12、シベリアンハムスター13またはヤク14のような他の動物からの一次セルトリ細胞の単離のために使用することができます。生殖細胞を低張性ショックを汚染大量の除去のための単離手順15の端部に用いることができます。成体ラットは、16精巣からこれはまた、セルトリ細胞の効率的な単離を可能にします。濃縮されたセルトリ細胞懸濁液は、 チョウセンアサガオ朝鮮朝顔アグルチニン17で被覆されたレクチン皿上懸濁液をプレーティングすることにより生殖細胞から分離することができます。唯一のセルトリ細胞といくつかの残留のPTCは、レクチンプレートに付着します。 主にラットからの一次セ​​ルトリ細胞培養物は、ホルモンに対する応答性を調査またはマウスのセルトリ細胞リチウムのような細胞株を確立するために最初に使用されていますTM4 18ね。この細胞株は、今日まで、百以上の研究で検討されています。翻訳アプローチではセルトリ細胞は、全身性の免疫抑制19せずに長期移植片生存のための異種のまたは同種異系膵島の同時移植のように共培養細胞や組織の免疫保護のために利用されてきました。体細胞、生殖細胞（セルトリ細胞-生殖細胞）20と相互作用21 -単離されたセルトリ細胞は、上皮間葉（PTCCセルトリ細胞）を研究するため、共培養実験に使用されてきました。最近、一次セルトリ細胞は、Toll様受容体の発現と炎症性サイトカインの分泌、ならびに非病原性および尿路病原性大腸菌による感染後のサイトカインの発現をもたらすシグナル伝達カスケードを調査するために使用されています大腸菌 22。他の最近の研究は、精巣の免疫Pを研究するためのセルトリ細胞を使用していましたrivilege 23およびテストステロン前処理は、LPS誘導性の炎症応答24を抑制することを実証しました。

<table border="0" cellpadding="0" cellspacing="0" width="813">
	<tbody>
		<tr height="80">
			<td height="80" style="height:80px;width:289px;">Actin (smooth muscle) antibody clone 1A4</td>
			<td style="width:111px;">Dako</td>
			<td style="width:124px;">M0851</td>
			<td style="width:289px;">Monoclonal mouse anti human antibody. 
			Use 1:100 dilution for immunofluorescence.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Albumin bovine fraction V 
			Standard grade, lyophilized</td>
			<td style="width:111px;">Serva</td>
			<td style="width:124px;">11930.04</td>
			<td style="width:289px;">Filter (0.45 &#181;m) BSA solutions used for immunofluorescence.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Corning&#8482; Cell Strainers 70&#160;&#181;m</td>
			<td style="width:111px;">Corning</td>
			<td style="width:124px;">431751</td>
			<td>White color</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Collagenase A from Clostridium histolyticum</td>
			<td style="width:111px;">Roche</td>
			<td style="width:124px;">10103586001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">DAPI mountant ProLong&#174; Gold Antifade</td>
			<td style="width:111px;">Life technologies</td>
			<td style="width:124px;">P-36931</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">D-glucose</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">G8644</td>
			<td>100 g/l</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Dulbecco&#180;s PBS without Ca2+/Mg2+</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">DNase I</td>
			<td style="width:111px;">Roche</td>
			<td style="width:124px;">10104159001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Electron microscope</td>
			<td style="width:111px;">Zeiss</td>
			<td>EM 109S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Fluorescence microscope</td>
			<td style="width:111px;">Zeiss</td>
			<td style="width:124px;">Axioplan 2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Hyaluronidase from bovine testis</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">H3506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Inverted light microscope</td>
			<td style="width:111px;">Olympus</td>
			<td style="width:124px;">CKX41</td>
			<td>Routine cell culture microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Mouse IgG / IgM (H+L) polyclonal 
			secondary Antibody&#160;</td>
			<td style="width:111px;">Life technologies</td>
			<td style="width:124px;">A-10684</td>
			<td>Alexa Fluor&#174; 488 conjugate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Oil Red O</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">O0625</td>
			<td style="width:289px;">Has replaced Sudan III and Sudan IV 
			because of brighter color.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Paraformaldehyde</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">P6148</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Penicillin (5,000&#160;U/ml)/ 
			Streptomycin (5,000 &#181;g/ml)</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">15070-063</td>
			<td>100x solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">RPMI-1640</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">21875-034</td>
			<td>Contains 300 mg/L L-glutamine</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Trypsin from porcine pancreas</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">T5266</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Trypan Blue Stain (0.4%)</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:289px;">Trypsin inhibitor from soybean</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">T6522</td>
			<td style="width:289px;">The Sigma product is considerably cheaper than the previously used 
			BPTI (Aprotinin) from Roche.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:289px;">Vimentin antibody (V9)</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">V6630</td>
			<td style="width:289px;">Monoclonal mouse anti pig vimentin antibody.&#160; Use 1:100 dilution for immunofluorescence.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Wistar WU rats</td>
			<td style="width:111px;">Charles River</td>
			<td style="width:124px;">N/A</td>
			<td>Should be 19 days old on day of experiment.</td>
		</tr>
	</tbody>
</table>

isolation of sertoli cells and peritubular cells from rat testes

分化した精子が最初思春期の時に表示されるので、精巣は、特に雄性配偶子に、独自の方法で免疫系に挑戦 - 十年以上全身性免疫寛容の確立後。精子形成細胞は、免疫系によって非自己として見ることができるタンパク質の数を表します。精巣は、その後、一方ではこれらのネオ抗原に対する寛容を確立することができるが、それでも一方、感染症や腫瘍の発生から自身を守ることができなければなりません。したがって、精巣は、有害な炎症性免疫応答を惹起することなく、外来抗原を容認し、体内のいくつかの免疫特権サイトの一つです。セルトリ細胞は精巣のこの免疫特権環境の維持のために重要な役割を果たし、また、外国の環境でcotransplanted細胞の生存を延長します。したがって、一次セルトリ細胞は、電子することはできません精巣の免疫特権を研究するための重要なツールですasily確立された細胞株または他の細胞モデルに置き換えられました。必要に応じて管周囲の細胞 - - ラットの精巣から一日以内にここでは、セルトリ細胞の単離のために詳細かつ包括的なプロトコルを提示します。

説明する手順では、10ラットの精巣から約12×10 7セルトリ細胞の単離を可能にします。 6〜7 6ウェルプレートは、7日目の実験のために利用可能であることをトリプシンDNアーゼI消化がセルトリ細胞の単離中に最も重要なステップになるように3×10 6細胞を6ウェルプレートにウェル当たり播種します。この時点での消化が遠く前進された場合に、生殖細胞...

Watch this Scientific Journal Video about Isolation of Sertoli Cells and Peritubular Cells from Rat Testes at JoVE.com

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

一次セルトリ細胞は精巣免疫特権、炎症または感染の間シグナル伝達、およびそれらの免疫防御特性の利用を研究するために必要とされます。ここでは、高度に精製された一次セルトリ細胞およびラットの精巣から管周囲の細胞を単離するための酵素ベースのプロトコルを記述します。

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-of-sertoli-cells-and-peritubular-cells-from-rat-testes

Research

JoVE Journal

Immunology and Infection

22.3K ビュー.  Justus-Liebig-Universität Gießen. 一次セルトリ細胞は精巣免疫特権、炎症または感染の間シグナル伝達、およびそれらの免疫防御特性の利用を研究するために必要とされます。ここでは、高度に精製された一次セルトリ細胞およびラットの精巣から管周囲の細胞を単離するための酵素ベースのプロトコルを記述します。 

ビデオ: Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

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 ...

免疫・感染学

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 ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

細胞運命を以下に<em&gt; E大腸菌の</emラムダファージによる感染後&gt;

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

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 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 Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

ヒト胸腺からの骨髄樹状細胞と上皮細胞の単離

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

腎臓からの二重否定αβT細胞の分離

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

ネズミリンパ節間質細胞の単離

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

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 ...