We thank Aaron Rae (Emory University Department of Pediatrics and Children's Healthcare of Atlanta Flow Core) for cell sorting. This work was supported by NIH grant AA01787001, a Career Development Award from the Crohn's and Colitis Foundation of America, and an Emory-Egleston Children's Research Center seed grant to T.L.D.

1。腸上皮細胞の解離および解離試薬や機器の調製： <ul><li>室温へのCa 2 + / Mg 2 +のフリーのPBS（CMF PBS）暖かい。 </li><li> 5％FBS（CMF HBSS / FBS）と室温に2mMのEDTAと暖かいのCa 2 + / Mg 2 +のフリーのHBSS。 </li><li> 37暖かいオービタルシェーカー℃、 </li></ul> 注：ステップ1.1から1.7までは、細胞死の程度を最小限に抑え、最大の細胞収率を達成するために、できるだけ早く実行する必要があります。 <ol><li>腹部と胸部の上にCO 2チャンバーとスプレー70％エタノールでマウスを安楽死させる。 </li><li>バックシザーと皮腹膜を露出する皮膚と腹部の真ん中に小さな水平方向の切開を行います。 </li><li>幽門括約筋で切断して上部小腸から胃を分離するために進んでください。ピンセットで腸間膜を離れていじめると、再びカット大腸から小腸全体を解放するために回腸盲腸弁。肛門縁で切断して、再度大腸が解放されるまで腸間膜を離れていじめる。 </li><li>縦方向にはさみを使用して大腸を開いてカットし、室温でCMF PBSで腸管腔から糞便の内容と粘液を洗い流してください。 </li><li>肉眼的に小腸の抗腸間膜表面に沿ってパイエル板を分析し、長手方向に小腸を開いてカットするはさみや鉗子を使用しています。 </li><li>室温で糞便の内容とCMF、PBS中の粘液の小腸内腔を洗浄します。 </li><li>別々に予め温めておいたCMF HBSS / FBSおよび2mM EDTA 30mlを含む別の50 mlコニカルチューブに約1.5cmの部分と所定の位置に大きい/小さい腸を切った。 50 mlコニカルチューブあたり1以上の腸を追加しないでください。 </li><li>水平オービタルシェーカーに各50 mlコニカルチューブを配置し、℃、&lt;37℃で20分間、250 rpmで振とう/ LI&gt; <li>廃棄物のバケットを介して単一のメッシュワイヤーストレーナーを配置し、腸の1.5cmの部分を回復することを介して各50 mlコニカルチューブの内容物を注ぐと2で予め温めておいたCHBSS / FBSの30ミリリットルを含む別の50 mlコニカルチューブに置くmMのEDTA。 </li><li> 1.8を繰り返します。 </li></ol> 2。組織の消化と腸細胞の単離試薬や機器の準備： <ul><li>コラゲナーゼ溶液： のDNase Iの40μg/ mlのであらかじめ温めておいたCMF HBSS / FBSに溶解した1.5​​ mg / mlのタイプVIIIのコラゲナーゼ</li><li>予め温めておいたCMF HBSS / FBSおよび2mM EDTA。 </li><li> 37℃で予め温めておいたオービタルシェーカー℃、 </li><li>氷冷CMF HBSS / FBS。 </li></ul><ol><li>揺れの第二ラウンドの後、ストレーナーを介して各50 mlコニカルチューブの内容物を注ぎ、小さなプラスチックに腸の1.5cmの部分を転送するペーパータオルを使用して、過剰なメディアを離れてたたく後、ボートの重量を量る。 </li><li>急速に1をミンチ腸の0.5センチメートル部分は重量のボートで直接はさみを使用して、コラゲナーゼ溶液20mlにミンチ腸を追加します。水平方向に37℃で10〜20分間、200 rpmでオービタルシェーカーとダイジェストに各50 mlコニカルチューブを配置℃で最適化に関する以下の説明を参照してください。 </li><li> 50mlコニカルチューブに直接100μmのセルストレーナーを介して任意の残りの腸組織、フィルタの徹底した解離を確保するために簡単にボルテックスします。 </li><li> CMF HBSS / FBSおよび4℃で5分間1500rpmで遠心分離℃でそれぞれ50 mlコニカルチューブオフトップ固体ペレットを遠心分離の後にコロンのサンプルで観察されていない場合、サンプルは3.5分間再度遠心分離する必要があります。もう一度、この洗浄ステップを繰り返します。 </li><li>上清を捨て、氷の上に氷冷CMF HBSS / FBSと場所をサンプル中の細胞ペレットを再懸濁します。 </li><li>高速セルsorti用の磁気ビーズ濃縮のためのFACS収集/分析または第4項については、セクション3に進みますNG。 </li></ol> 3。樹状細胞とマクロファージのマルチカラーフローサイトメトリー分析のための抗体染色試薬や機器の準備： <ul><li>氷冷PBS CMF。 </li><li>氷冷染色緩衝液（CMF PBS + 5％FBS）。 </li><li> LIVE / DEAD修正可能アクア死細胞染色キットを使用して、1:1000希釈で氷冷CMF PBSで染色死細胞を準備します。 </li><li> CD45-PerCPで、CD103-PEは、CD11c-APC、MHC-II（IA B）のAlexa Fluor 700、CD11bを以下の蛍光標識モノクローナル抗体（mAb）の氷冷染色バッファーに追加することによって、抗体染色カクテルを準備します。 -eFluor 450 F4/80-PE-Cy7。 </li></ul><ol><li> 5 mlのポリスチレン丸底（FACS）チューブに細胞を移す。 </li><li>氷冷CMF PBSで2回細胞を洗浄します。 </li><li>死んだ細胞とインキュベートしたサンプルは、暗所で氷上で15分間染色する。 </li><li>氷冷CMF PBSで2回細胞を洗浄します。 </li><li>氷冷染色で2.4G2anti-FcγRIII/IIで細胞をブロックする氷上で10分間バッファ。 </li><li>氷冷染色緩衝液で細胞を洗浄します。 </li><li>暗闇の中で氷上で20分間、抗体染色のカクテルでサンプルをインキュベートします。 </li><li>回氷冷染色緩衝液で細胞を洗浄し、氷冷染色バッファー400μlのサンプルを再懸濁し、FACSチューブに40μmのフィルターキャップを通過します。 </li><li>第5節、 図1にゲート戦略で定義されているLSR IIフローサイトメーター（BD）のサンプルを取得します。 </li></ol> 4。腸からの樹状細胞とマクロファージの濃縮試薬や機器の準備： <ul><li>氷冷染色緩衝液（CMF PBS + 5％FBS）。 </li></ul><ol><li>製造元の指示に従って、CD11bをおよびCD11c MACSビーズでステップ2.5から得られた単一細胞懸濁液をインキュベートします。 </li><li>遠心分離に続いて氷冷染色緩衝液で細胞を洗浄します。 </li><li>上清を捨て、1mlの氷冷染色バッファーで細胞ペレットを再懸濁40μmのセルストレーナーに続いて100μmのセルストレーナーを通過します。 </li><li> MACS LS磁気カラムを用いて、ポジティブセレクションにより磁気ビーズ付着細胞を濃縮。 </li><li>ステップ4.2を繰り返し、上清を捨てる。 </li><li> 、ステップ3.7で説明表面マーカーモノクローナル抗体で細胞をインキュベートします。 </li><li>氷冷染色緩衝液で2回磁気ビーズ濃縮細胞を洗浄します。アジ化ナトリウムをせずに500μLの氷冷染色バッファーで細胞ペレットを再懸濁し、そしてFACSチューブに40μmのセルストレーナーを通過します。 </li><li>腸のDCおよび/または興味のあるマクロファージサブセットをソートするためにBD ARIA IIセルソーターでソーティングFACSに進みます。 </li></ol> 5。 LP APCのためのゲーティング戦略 注 ：染色腸の細胞は、正と負の集団を分離するためにゲートの適切な配置を支援するために、ネガティブコントロールとして利用することができるのでご注意ください。 <ol><li> 図に示すように、1、ドットプロットを作成し、二重のイベントの除外（ 図1BおよびC）に続いて染色死細胞（ 図1A）のために陽性の細胞を除外します。その後、前方および側方散乱は（ 図1D）の残骸を除外することを確認することに応じて目的の細胞のゲート。 </li><li> CD45上の別のドットプロットし、さらにゲートを作成して+とIA B +細胞は、表現型APCを特徴づける（ 図1E）。 </li><li>独立したドットプロットで、特定のDCおよびマクロファージサブセット（;図1F R1、R2、およびR3）を区別するために、CD11bをおよびCD11c発現について分析します。 R1の細胞は、CD11cはある+ CD11bを鈍い/ -細胞。地域、R2はまた、CD11bを発現し、R1がR2の細胞の細胞のように、これらの細胞はCD11cの表面発現の同様のレベルを持っていることをそのような線引きされています。 -その後、R3領域はCD11bの+と/鈍いのCD11cているセルに指定されています。 </li><li>不定冠詞領域R1、R2、およびそれぞれ、マクロファージやDC集団を区別するために、F4/80とCD103発現のためにR3をさらにalyze。のCD11c + CD11bを鈍い/ - R1急行αEインテグリン高レベルのCD103とF4/80の低レベル（ 図1G）の細胞。 CD11bを+のCD11c + R2の細胞がCD103とF4/80（ 図1H）彼らの二分式に基づいて樹状細胞やマクロファージの両方で構成されています。最後に、CD11bを+ CD11cは鈍い/ - R3の細胞は、F4/80 +とCD103の表現型のプロファイルに基づいてマクロファージを構成する- （ 図1I）16。 </li></ol> 6。代表的な結果<img alt="図1" src="/files/ftp_upload/4040/4040fig1.jpg" /> 図1。腸の樹状細胞やマクロファージのゲーティング戦略。死細胞（A）とダブレット（BおよびC）は、最初の分析から除外して、小さなintestiた内部の細胞は、転送することがそれに応じてゲートされ、側方散乱（D）と、APCはCD45のように定義されたIA + B +（E）。マクロファージや樹状細胞はCD11bをおよびCD11c（F）の発現により同定した。 R1（G）、R2（H）とR3（I）個体群の事前ゲートセルのCD103とF4/80の発現を解析した。 <img alt="図2" src="/files/ftp_upload/4040/4040fig2.jpg" /> 図2。細胞収率と抗体染色の質は消化時間に依存します。CD11bをおよびCD11c染色パターンと下の（A、D）の全細胞収量、最適な-（B、E）以上消化し ​​ます（C、F）腸組織。 腸の細胞は、BD LSR IIのFACSにより分析したC57BL / 6マウス小腸および樹状細胞やマクロファージから単離した。電圧と補償は染色、単一の蛍光色素で染色した脾細胞を使用して設定された。死んだ細胞（ 図1A）とダブレットた（ 図1BおよびC）は最初から除外された分析。興味のある細胞は、その後に転送するようによると、側方散乱（ 図1D）CD45ゲーティングに続いて+とIA B +細胞（ 図1E）を分析した。その後、CD11bをおよびCD11cの発現がCD45の間で評価されたIA + bの 3つの領域を（R1、R2、およびR3。図1F）線引きする+細胞。 3地域におけるCD103とF4/80の発現は、それぞれ、樹状細胞やマクロファージを区別するために評価した。のCD11c + CD11bを鈍い/ - R1の細胞は、インテグリンαE、CD103、およびF4/80の低レベル（ 図1G）の高いレベルを表明した。 CD11bを+のCD11c + CD11bを+のCD11c /鈍いながらR2の細胞は、CD103とF4/80（ 図1H）彼らの二分式に基づいて樹状細胞やマクロファージの両方で構成されていた- R3の細胞はF4 /の表現型プロファイルに基づいてマクロファージを構成する80 +とCD103 - （ <strオング&gt;図。 1I）16。 R3ゲートにR2ゲートとマクロファージ内マクロファージは、同様の前方および側方散乱特性を持っているおよびCD11c発現により区別できます。これらのサブセットの機能的な二分法は、完全には理解のままです。 全細胞収量およびCD11bおよびCD11cの発現に及ぼす組織の消化時間の関係を図2に示されています。 3分（下の消化）のために消化し ​​た腸組織は、低総細胞数（ 図2D）を得たと特性（ 図2A）のために利用できるため、いくつかの樹状細胞やマクロファージ。 11分のための組織消化では、CD11bをおよびCD11cの高レベルで発現した（ 図2C）表現型が異なっていた樹状細胞とマクロファージの集団で生きている細胞（ 図2E）の堅牢な収量を生産した。対照的に、50分（以上消化）の消化は、同様の細胞収率をもたらした時のCO最適化された消化（ 図2E及びF）にmparedしかしながら、CD11bをおよびCD11cを使用して異なる細胞集団の描写（ 図2C）CD11cは減少の発現など、よりあいまいになり、死んだ細胞の数は、（データは示さず）増加しました。

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Immunol. 9, 769-776 (2008).</li><li>Schenk, M., Bouchon, A., Seibold, F., Mueller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TREM-1--expressing+intestinal+macrophages+crucially+amplify+chronic+inflammation+in+experimental+colitis+and+inflammatory+bowel+diseases.">TREM-1--expressing intestinal macrophages crucially amplify chronic inflammation in experimental colitis and inflammatory bowel diseases.</a> J. Clin. Invest. 117, 3097-3106 (2007).</li><li>Sun, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+intestine+lamina+propria+dendritic+cells+promote+de+novo+generation+of+Foxp3+T+reg+cells+via+retinoic+acid.">Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 T reg cells via retinoic acid.</a> J. Exp. Med. 204, 1775-1785 (2007).</li><li>Kamada, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormally+differentiated+subsets+of+intestinal+macrophage+play+a+key+role+in+Th1-dominant+chronic+colitis+through+excess+production+of+IL-12+and+IL-23+in+response+to+bacteria.">Abnormally differentiated subsets of intestinal macrophage play a key role in Th1-dominant chronic colitis through excess production of IL-12 and IL-23 in response to bacteria.</a> J. Immunol. 175, 6900-6908 (2005).</li><li>Denning, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++Th+cells+resembling+regulatory+T+cells+that+inhibit+chronic+colitis+differentiate+in+the+absence+of+interactions+between+CD4+and+class+II+MHC.">CD4+ Th cells resembling regulatory T cells that inhibit chronic colitis differentiate in the absence of interactions between CD4 and class II MHC.</a> J. Immunol. 171, 2279-2286 (2003).</li></ol>

利害の衝突が宣言されません。

<img src="/files/ftp_upload/4040/4040fig3.jpg" alt="図3"/> 図3。細胞収率と表面抗原の発現を最適化するための重要な要素。細胞収率と表面抗原の発現を直接組織消化の期間に影響される、コラゲナーゼの固有の特性、ミンチ組織の程度、炎症の有無、これは、組織の整合性と細胞性に影響を与える可能性があります。不十分な組織の消化は、分析のための細胞の不足につながるかもしれ?...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the Reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>1X PBS, Ca2+- and Mg2+-free</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hank's balanced salt solution (HBSS) with phenol red</td>
 <td>Fisher Scientific</td>
 <td>SH3001603</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium bicarbonate</td>
 <td>Sigma</td>
 <td>S6014</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1M HEPES in 0.85% NaCl</td>
 <td>Lonza</td>
 <td>17-737E</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal bovine serum (FBS)</td>
 <td>Atlanta biologicals</td>
 <td>S11150H</td>
 <td>Heat-inactivated</td>
 </tr>
 <tr>
 <td>0.5M EDTA (pH 8.0)</td>
 <td>Cellgro</td>
 <td>46-034-CI</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Collagenase type VIII </td>
 <td>Sigma</td>
 <td>C2139</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DNase I</td>
 <td>Roche</td>
 <td>14785000</td>
 <td>Stock solution: 100mg/ml</td>
 </tr>
 <tr>
 <td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit for 405 nm excitation</td>
 <td>Invitrogen</td>
 <td>L34957</td>
 <td>Use at 1:1000</td>
 </tr>
 <tr>
 <td>CD45-PerCP mAb (30F11)</td>
 <td>BD</td>
 <td>557235</td>
 <td>Use at 1:100</td>
 </tr>
 <tr>
 <td>CD103-PE mAb (M290)</td>
 <td>BD</td>
 <td>557495</td>
 <td>Use at 1:100</td>
 </tr>
 <tr>
 <td>Fc&#x03B3;RIII/II mAb (2.4G2)</td>
 <td>BD</td>
 <td>553141</td>
 <td>Use at 1:200</td>
 </tr>
 <tr>
 <td>CD11c-APC mAb (N418)</td>
 <td>eBioscience</td>
 <td>17-0114-82</td>
 <td>Use at 1:100</td>
 </tr>
 <tr>
 <td>MHC-II (I-Ab)-Alexa Fluor 700 mAb </td>
 <td>eBioscience</td>
 <td>56-5321-82</td>
 <td>Use at 1:100</td>
 </tr>
 <tr>
 <td>CD11b-eFluor 450 mAb (M1/70)</td>
 <td>eBioscience</td>
 <td>48-0112-82</td>
 <td>Use at 1:200 </td>
 </tr>
 <tr>
 <td>F4/80-PE-Cy7 mAb (BM8)</td>
 <td>eBioscience</td>
 <td>25-4801-82</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CD11b microbeads</td>
 <td>Miltenyi Biotec</td>
 <td>130-049-601</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CD11c microbeads</td>
 <td>Miltenyi Biotec</td>
 <td>130-052-001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 mL conical tubes</td>
 <td>BD Falcon</td>
 <td>352098</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Single mesh wire strainer</td>
 <td>Chefmate</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Small weigh boat</td>
 <td>Fisher Scientific</td>
 <td>08-732-116</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>100 &mu;m cell strainer</td>
 <td>BD Falcon</td>
 <td>352360</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>40 &mu;m cell strainer</td>
 <td>BD Falcon</td>
 <td>352340</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>5 mL polystyrene round-bottom tubes</td>
 <td>BD Falcon</td>
 <td>352235</td>
 <td>Use at 1:100</td>
 </tr>
 <tr>
 <td>MaxQ 4450 benchtop orbital shaker</td>
 <td>Thermo Scientific</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>LS MACS column</td>
 <td>Miltenyi Biotec</td>
 <td>130-042-401</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>LSR II</td>
 <td>BD</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FACSAria II</td>
 <td>BD</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

isolation and characterization of dendritic cells and macrophages from the mouse intestine

腸内に付随して残りの侵襲的な病原体1,2に向かって炎症反応をマウントする態勢を整えながら、共生植物や食物抗原に対する寛容の促進に関与している自然免疫と適応免疫細胞のユニークな集団を常駐します。抗原提示細胞、特に樹状細胞やマクロファージは、感知し、適切に細菌3月14日に応答する能力を介して腸管免疫恒常性を維持する上で重要な役割を果たしている。腸の樹状細胞とマクロファージの効率的な分離は、これらの細胞の表現型と機能を特徴づける重要なステップです。樹状細胞とマクロファージを含む腸管免疫細胞を、単離の多くの効果的な方法は6,10,15-24を説明してきたが、多くは否定的な細胞表面抗原の発現、細胞の生存、および/ ​​または細胞収量に影響を与える可能性がある長い消化時間に依存しています。ここでは、詳細viabl大量の迅速分離するための方法論をE、腸の樹状細胞やマクロファージ。腸の樹状細胞とマクロファージの表現型の特性が直接マルチカラーフローサイトメトリー分析のために特定の蛍光標識モノクローナル抗体を用いて分離された腸の細胞を染色することにより行われる。さらに、高純度のDCおよびマクロファージの集団は細胞選別が続くとCD11b CD11cは磁気活性化細胞選別ビーズを利用した機能性研究のために隔離されています。

Watch this Scientific Journal Video about Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine at JoVE.com

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

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-characterization-dendritic-cells-macrophages-from-mouse

Research

JoVE Journal

Immunology and Infection

38.9K ビュー.  Emory University. 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.

ビデオ: Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

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 ...

免疫・感染学

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 ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

T細胞は、樹状細胞からTransinfectionによって細菌をキャプチャ

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

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 ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

マウスの骨髄からGM-CSF由来肺胞のようなマクロファージや樹状細胞の生成および同定

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

イメージングフローサイトメトリーによるヒト単球由来樹状細胞の特徴付け：二つの単球の分離プロトコルの比較

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

フローサイトメトリーによるラットの腎臓からのマクロファージの表現型の特徴

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

マウスの骨髄から形質細胞様樹状細胞の精製のために、蛍光活性化細胞選別

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

全血からのヒト単球由来樹状細胞の生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.