著者らは、顕微鏡との彼の援助のための細胞選別とピーターモットラムと彼女の助けのためにLSUのフローサイトメトリーコアファシリティーでマリリンディートリッヒに感謝したいと思います。この作品は、フライトアテンダント医学研究所、LSU -競争的研究プログラム賞、およびNIH / NIAID助成金P20 RR020159とR03AI081171によって資金を供給された。

1。肺血流と、単一の細胞懸濁液<ol><li>大腿静脈を経由してケタミン/キシラジン麻酔の混合物（1mg /マウスケタミン1.8では、キシラジン0.19）と放血の腹腔内注射したマウスを安楽死させる</li><li>カットと軽く腹膜の外側の皮を引き戻すことによって胸腔を公開。心臓と肺の両方を公開するために、胸郭を切断してダイアフラムを開くに進みます。 </li><li> EDTA - HBSS（カルシウム/マグネシウムを含まないHBSSで1mMのEDTA）を充填した5mlの注射器を使って静かに肺を灌流。 25 Gの針を接続し、右心室に針を挿入する。注入時の漏れを防ぐために、鉗子を使用して針の周囲心筋組織を固定します。一定の圧力を維持しながらゆっくりとソリューションを注入する。正確な血流は肺の膨張とピンク/白への色の変化になります。 </li><li>この組織からの潜在的な汚染を破棄するように所属リンパ節を削除します。 </li><li>収集肺組織では、氷の上でペトリ皿に移し、カミソリの刃を使用して、小さな断片に切り刻む。 </li><li>を5 ml /コラゲナーゼ消化液の肺を含むgentleMACS C管に接地された組織を転送する（コラゲナーゼタイプ1Aは0.5 mg / mlのプラスIV型ウシ膵臓のDNase 20 5％FBS、100 U / mlペニシリンおよび100μg/mlを含むHBSSでμg/ mlの/ mlのストレプトマイシン）。 </li><li>単一の細胞懸濁液を得るためにgentleMACSのdissociatorを使用してください。 m_lung_01：プログラムを選択してください。 30分間37℃でサンプルをインキュベートする。組織片を懸濁するためにチューブを5分ごとに振る。プログラムm_lung_02に進みます。 </li><li> 4で335 x gで10分間、15mlコニカルチューブと遠心分離機にサンプルを移す℃にこのステップの後、4で全ての試薬と遠心分離を維持° Cは、細胞の生存率の低下を防止する。 </li><li>上清を捨て、室温で1分間ACK溶解緩衝液2 mlを加え、残りの赤血球を溶解する。 13で細胞を洗浄4で335 x gで10分間、冷たいPBS/0.5％BSAおよび遠心℃のミリリットル</li><li>コー​​ルドPBS/0.5％のBSAの5mlに上清、再懸合計セルを廃棄し、100μmのナイロンメッシュを徹底的に渡します。 </li><li>トリパンブルー排除色素を使用して、総細胞数を決定します。 </li></ol> 2。磁気分離とCD11c +細胞の濃縮<ol><li> 4℃で200 × gで10分間、単一の細胞懸濁液を遠心し、上清を捨てる。 </li><li> 10 8合計のセルごとに分離バッファー400μlの再懸濁し細胞ペレット（PBS、0.5％BSA、および2mM EDTA）。 4℃で30分間anti-CD16/CD32抗体（0.5μg/ 10 6細胞）を使用して、結合ブロックのFcを介する非特異的抗体℃に</li><li> 4で200 × gで10分間冷分離バッファと遠心を10ml℃で細胞を洗浄分離バッファー400μlで細胞を再懸濁します。 </li><li> 10 8あたりのCD11cマイクロビーズを100μlを加え</SUP&gt;の合計のセルを。よく混ぜ、冷蔵庫で15分（2〜8℃）インキュベートする。 </li><li> 4℃で200 × gで10分間冷分離バッファと遠心の10mlで細胞を洗浄し、上清を捨てる。 </li><li>冷分離緩衝液3mlで細胞ペレットを再懸濁します。 </li><li>プログラムのposseldを選択することにより、autoMACS ProのTMのセパレータを使用して磁気分離を実行します。陽性画分（0.5 ml）を収集する。 </li></ol> 3。従来の樹状細胞（CDC）の分離<ol><li>抗CD11c PE - Cy7とanti-IA/IE（MHC - II）- FITC抗体で30分間、4℃でインキュベートし、濃縮CD11c陽性細胞懸濁液抗体の最適化された濃度は、細胞の0.5μg/ 10 6です。 </li><li> 4で335 x gで10分間、冷たいPBS/0.5％BSAおよび遠心℃で細胞を洗浄再懸PBS/0.5％BSAを0.5mlの中の細胞と40μmのナイロンメッシュを介してそれらを渡す。 </li><li>に進んでください純度のモードでFACSのアリアのセルソーターを使用してセルを並べ替える。精製された細胞の損傷を防止するためにコレクションチューブにPBS/0.5％BSAを100μlのを追加。細胞選別のプロセスを通して冷蔵温度であなたの細胞を保つ。 </li></ol> 4。単一の細胞懸濁液およびCD11c +細胞の濃縮を取得するための代替プロトコル<ol><li> gentleMACSのdissociatorの交換では、ステップ1.5から接地肺組織は、37℃で1時間肺とインキュベートあたりのコラゲナーゼ溶液5 mlを入れた15 mlコニカルチューブに移すことができる渦は、細胞が15分ごとに組織断片を再懸濁するため。 inとout 20 Gの針に接続して、3 mlの注射器を介してサンプルを6-8回を渡すことによって、組織を混乱させる。プロセス中に泡を避ける、特に細胞の生存率が低下します。単一の細胞懸濁液が得られれば、1.8に進みます。 </li><li> AutoMACSPro TM separaの交換でTorは、樹状細胞の細胞の濃縮のための磁気分離は、また、2つのMACSカラムを介して手動で細胞懸濁液を渡すことで実行できます。適切な列の選択は、サンプルサイズが異なります。 </li></ol> 5。代表的な結果： 肺CDCは、CD11cはハイ / MHC - II ハイの細胞集団として同定されています。図に示す。 1は、CDCは、脾臓（4.8％）などのいくつかの他の組織に比べて1.4％の小さな割合を表しています。しかし、脾臓とは対照的に、肺のCD11c陽性細胞は、CDCとは異なる細胞集団を含む、MHC - IIの異なる量を表現する。単一細胞調製後、全肺の細胞の細胞収率はわずか1％以上がCDCのサブセットを表す、そこから60〜70パーセントの生存率と3.8から3.0程度× 10 7細胞/肺であった。この割合は、総肺CDCは約10 tをインクリメントされた樹状細胞の磁気分離後に増加したオリジナルの準備（図2A）からのIME（&gt; 16％）。しかし、MHC - II（CD11cはHI / MHC - II LO、マクロファージ）の少量を表現するCD11cは細胞が肺CDCのサブセットと混合され、高汚染の細胞集団（&gt; 70％）を表す。図に示す。 2B、それらの細胞は、CDCの純度は&gt; 96％に達した細胞選別工程後に除去した。全体の手順の後にCDCの通常の収率は5 × 10 4細胞/肺であった。顕微鏡写真は、代表的な樹状突起（上のパネル）と丸い細胞としてCDCの形態的特徴を示す。一方、CD11cはハイ / MHC - II LOは、マクロファージ（Mφ、下のパネル）12の典型的な細胞の突起を示す形態学的に異なるサブセットを表す。 <img alt="図1" src="/files/ftp_upload/3563/3563fig1.jpg" /> マウスの肺及び脾臓で図1の差動DC周波数 。肺C57BL / 6マウスからNDの脾臓組織をコラゲナーゼで収集し、処理した。コラゲナーゼ消化後、細胞を抗マウスCD11cは- PE - Cy7および抗マウスIA / IE - FITCで染色した。代表的なフローサイトメトリープロットでは、肺と脾臓におけるCDCの割合（CD11cはHI / MHC - II ハイ ） を示す。 <img alt="図2" src="/files/ftp_upload/3563/3563fig2.jpg" /> （2）マウスの肺からCDCの分離。マウスの肺の単一細胞の懸濁液を抗CD11cマイクロビーズで標識し、自動細胞分離装置を通過した。）代表的なプロットは、CDCの割合を示す図 （樹状細胞ハイ / MHC - II HI）とマクロファージ（Mφ、CD11cはハイ / MHC - II LO）CD11cは濃縮後の集団。濃縮分画のさらなる染色は、抗マウスCD11cは- PE - Cy7および抗マウスIA / IE - FITCを使用して続く。二重陽性細胞をソートし、フローサイトメトリーによって分析した。へのその形態を特定する、セルが変更されたライト-ギムザ染色でサイトスピンし、染色した。B）代表プロットは、細胞選別後のCDCおよびMφの割合を示す。顕微鏡写真では肺CDCと肺のMφの代表画像を示す。バー=20μmのスケール。

<ol>
	<li>Pulendran, B., Palucka, K., Banchereau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+pathogens+and+tuning+immune+responses.">Sensing pathogens and tuning immune responses.</a> Science. 293, 253-256 (2001).</li><li>Banchereau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunobiology+of+dendritic+cells.">Immunobiology of dendritic cells.</a> Annual Review of Immunology. 18, 767-811 (2000).</li><li>Manicassamy, S., Pulendran, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+control+of+tolerogenic+responses.">Dendritic cell control of tolerogenic responses.</a> Immunol. Rev. 241, 206-227 (2011).</li><li>Steinman, R. M., Cohn, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+cell+type+in+peripheral+lymphoid+organs+of+mice.+I.+Morphology,+quantitation,+tissue+distribution.">Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution.</a> J. Exp. Med. 137, 1142-1162 (1973).</li><li>Asselin-Paturel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+type+I+IFN-producing+cells+are+immature+APCs+with+plasmacytoid+morphology.">Mouse type I IFN-producing cells are immature APCs with plasmacytoid morphology.</a> Nat. Immunol. 2, 1144-1150 (2001).</li><li>Chan, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-producing+killer+dendritic+cells+provide+a+link+between+innate+and+adaptive+immunity.">Interferon-producing killer dendritic cells provide a link between innate and adaptive immunity.</a> Nat. Med. 12, 207-213 (2006).</li><li>Taieb, 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+novel+dendritic+cell+subset+involved+in+tumor+immunosurveillance.">A novel dendritic cell subset involved in tumor immunosurveillance.</a> Nat. Med. 12, 214-219 (2006).</li><li>Guerrero-Plata, A., Kolli, D., Hong, C., Casola, A., Garofalo, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subversion+of+pulmonary+dendritic+cell+function+by+paramyxovirus+infections.">Subversion of pulmonary dendritic cell function by paramyxovirus infections.</a> Journal of Immunology. 182, 3072-3083 (2009).</li><li>Larson, R. S., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+leukocyte+integrins.">Structure and function of leukocyte integrins.</a> Immunol. Rev. 114, 181-217 (1990).</li><li>Sung, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+major+lung+CD103+(alphaE)-beta7+integrin-positive+epithelial+dendritic+cell+population+expressing+Langerin+and+tight+junction+proteins.">A major lung CD103 (alphaE)-beta7 integrin-positive epithelial dendritic cell population expressing Langerin and tight junction proteins.</a> J. Immunol. 176, 2161-2172 (2006).</li><li>Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+CD11c++MHC+class+II-+precursors+generate+lung+dendritic+cells+during+respiratory+viral+infection,+but+are+depleted+in+the+process.">Local CD11c+ MHC class II- precursors generate lung dendritic cells during respiratory viral infection, but are depleted in the process.</a> J. Immunol. 177, 2536-2542 (2006).</li><li>Bhatia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+host+defense+against+Aspergillus+fumigatus+involves+alveolar+macrophages+with+a+predominance+of+alternatively+activated+phenotype.">Rapid host defense against Aspergillus fumigatus involves alveolar macrophages with a predominance of alternatively activated phenotype.</a> PLoS One. 6, e15943-e15943 (2011).</li><li>Shao, Z., Makinde, T. O., McGee, H. S., Wang, X., Agrawal, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fms-like+tyrosine+kinase+3+ligand+regulates+migratory+pattern+and+antigen+uptake+of+lung+dendritic+cell+subsets+in+a+murine+model+of+allergic+airway+inflammation.">Fms-like tyrosine kinase 3 ligand regulates migratory pattern and antigen uptake of lung dendritic cell subsets in a murine model of allergic airway inflammation.</a> J. Immunol. 183, 7531-75381 (2009).</li><li>Hao, X., Kim, T. S., Braciale, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+response+of+respiratory+dendritic+cell+subsets+to+influenza+virus+infection.">Differential response of respiratory dendritic cell subsets to influenza virus infection.</a> J. Virol. 82, 4908-4919 (2008).</li></ol>

著者らは、開示することは何もない。

肺マウスDCの分離は、呼吸器の刺激の広い範囲の研究のための重要なテクニックです。これらの細胞を得る工程は、細胞の喪失だけでなく、細胞の生存と純度を防ぐための重要な手順が含まれています。コレクションの前に肺を灌流しても、末梢細胞を排除するだけでなく、汚染物質の赤血球を削減することができます。肺の多数が処理されるときに自動化された解離の使用はadvanteogusことが...

<table width="200" border="1"><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（省略可能） </td></tr><tr><td> ACK溶解緩衝液</td><td>インビトロジェン</td><td> D6 - 0005DG </td><td></td></tr><tr><td>抗マウスのCD11c（HL3） </td><td> BD Pharmingen社</td><td> 5580979 </td><td> PE - Cy7共役</td></tr><tr><td>抗マウスIA / IE（269） </td><td> BD - Pharmingen社</td><td> 553623 </td><td>共役FITC </td></tr><tr><td> Collangenaseタイプ1A </td><td>シグマ</td><td> 9891 - 500mgの</td><td></td></tr><tr><td>セルストレーナー</td><td> BDファルコン</td><td> 352340、352360 </td><td></td></tr><tr><td> CD11cは（N418）マイクロビーズ</td><td>国内でのみ有効</td><td> 130-052-001 </td><td></td></tr><tr><td> DNase Iを</td><td>シグマ</td><td> D5025 - 150KU </td><td></td></tr><tr><td>ハンクス平衡塩類溶液</td><td>インビトロジェン</td><td> 14170 </td><td></td></tr><tr><td> HEPES緩衝液</td><td>インビトロジェン</td><td> 15630 </td><td></td></tr><tr><td>ペトリ皿は60mm </td><td> BDファルコン</td><td> 351016 </td><td></td></tr><tr><td> GentleMACS™Cチューブ</td><td>国内でのみ有効</td><td> 130-093-237 </td><td></td></tr><tr><td>ジェントルMACS dissociator </td><td> Miltentyi </td><td> 130-093-235 </td><td></td></tr><tr><td> AutoMACS - Pro™の</td><td>国内でのみ有効</td><td> 130-092-545 </td><td></td></tr><tr><td> FASCSアリア</td><td> BD </td><td></td><td></td></tr></table>

isolation of mouse lung dendritic cells

肺の樹状細胞（DC）は、侵入病原体の1,2と同様に気道の免疫寛容の回答3の制御でのセンシングにおいて重要な役割を果たします。肺の樹状細胞の少なくとも3つの主要なサブセットは、マウスで記載されている：従来のDC（CDC）4、形質DC（PDC）5とIFN -産生キラーDC（IKDC）6,7。 CDCのサブセットは、肺8で最も顕著なDCのサブセットです。 DCサブセットを識別するために知られている一般的なマーカーは、CD11cは、I型も単球、マクロファージ、好中球およびいくつかのB細胞9上に発現される膜貫通インテグリン（β2）です。一部の組織では、マウスDCを識別するマーカーとして樹状細胞を使用すると、ほとんどのCD11c +細胞は主要組織適合遺伝子複合体クラスII（MHC - II）の高レベルを表すCDCのサブセットを表す脾臓、のように、有効です。しかし、肺はものより異質な組織であるサイドDCサブセットは、MHC - IIの低レベルの試合CD11cは高レベルの表現の異なる細胞集団の割合が高いがあります。その特性上、ほとんどがF4/80、脾臓マクロファージのマーカーのその式に基づいて、CD11cはハイ MHC - II LOの肺の細胞集団は、潜在的なDC前駆11として、肺のマクロファージ、最近10として同定されている。 マウスPDCには対照的に、肺の免疫応答におけるCDCの特定の役割の研究は、これらの細胞の分離に役立つ可能性が特異的なマーカーの不足により制限されている。したがって、この作品では、我々は高度に精製されたマウスの肺CDCを分離する手順を説明します。肺DCサブセットの分離は、呼吸器病原体だけでなく、肺の宿主免疫応答を誘発する環境要因に応答してこれらの細胞の機能に関する洞察を得るために非常に便利なツールを表します。

Watch this Scientific Journal Video about Isolation of Mouse Lung Dendritic Cells at JoVE.com

Isolation of Mouse Lung Dendritic Cells

マウス肺樹状細胞の高度に精製された調製物が記載されている。特定の重点は、従来の樹状細胞サブセットの分離に与えられます。

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 ...

Research

JoVE Journal

Immunology and Infection

28.9K ビュー.  Louisiana State University. マウス肺樹状細胞の高度に精製された調製物が記載されている。特定の重点は、従来の樹状細胞サブセットの分離に与えられます。

ビデオ: Isolation of Mouse Lung Dendritic Cells

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 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 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 ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

マウスの樹状細胞サブセットの単離のための効率的かつ高収率方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

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 ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

レジオネラ・ニューモ外膜小胞：マクロファージ上における炎症誘発ポテンシャルの単離と解析

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

免疫組織化学およびフローサイトメトリーを用いた気管支生検における空間分布と表現型の同定：ヒト肺樹状細胞

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

精密カットライブ肺樹状細胞を可視化するために、マウスの肺スライスを

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

人間の女性の生殖器官から樹状細胞の表現型および機能研究の分離

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...