私たちは、博士に感謝しています。試薬を提供するためのマルクス・マンツとアービングワイズマン。我々はまた、それぞれ、フローサイトメトリー分析によって提供されるサービスとファーストと医学の国立台湾大学で第二のコア研究所とNTU病院の細胞選別基盤施設を認めます。この作品は、科学技術省、台湾（MOST 102から2320-B-002から030-MY3）と国民健康研究所、台湾（NHRI-EX102-10256SIとNHRI-EX103-10256SI）によってサポートされていました。

C57BL / 6野生型マウスは、国立実験動物センター（NLAC）、台湾から購入しました。すべてのマウスは、特定病原体を含まない条件下で飼育し、維持しました。プロトコルおよび動物使用手順を見直し、医学の国立台湾大学（許可番号：20120075）の施設内動物管理使用委員会によって承認されました。実験を行っている間に加えて、研究者は、動物のすべての痛みの可能性を減らすため、苦痛、または苦痛を行いました。手袋を着用して説明されているすべての手順は室温で行いました。 AC-6フィーダー細胞の調製注：（I.ワイズマン、スタンフォード大学によって提供された）AC-6.21（AC-6要するに）間質細胞ライン18は、RPMI中で維持されるべきである15％の熱不活性化ウシ胎児血清（FBS）を補充しました。フィーダー細胞として機能するには、AC-6細胞は、γ線を照射している3000ラド（30Gy）1日との共培養の前に、その増殖を防ぐために。注目メンテナンス中に過密放置すれば、AC-6の細胞は、その分化支持能を失う傾向がある、またはその通過数は20を超えている場合。 <ol><li> 1ミリリットルダルベッコのリン酸緩衝生理食塩水（DPBS）と吸引によってDPBSを削除すると1回洗浄AC-6細胞。 37 O℃で3~5分間、0.8 mlのトリプシン溶液（0.05％トリプシンおよびDPBSで0.5mMのEDTA）とAC-6細胞を、5％CO 2を処理した後、培養液の10容量にして、それを希釈することによって反応を停止させます単一細胞懸濁液を作ります。 </li><li> 5.9x10のAC-6細胞/ウェルを12ウェルプレートに4細胞をシードし、37°C ​​の 、5％CO 2、O ​​/ Nでインキュベートします。 注意：AC-6細胞の密度はのCDC生成を有利に働く低密度として非常に重要です。 4細胞/ウェル5.9x10の密度で播種は、AC-6のCLPからのpDCの導出に最適である次の日、によって密集度に到達することができます。 </li><li> γ-照射を使用して3000ラド（30グレイ）のAC-6細胞を照射します。 注意：AC-6を照射するために使用される用量は、増殖から細胞を防ぎ、しかものCLPからのDCの分化に必要なサイトカインおよび細胞 - 細胞接触を提供するのに十分に長い生存を保つために最適化されます。 </li><li>培地を吸引し、1mlの完全RPMI（10％熱不活性化FBS、50μg/ mlのゲンタマイシン、βメルカプトエタノール、50μMを含むRPMI）と交換してください。 </li></ol>マウスから2アイソBM細胞<ol><li> CO 2窒息と頸椎脱臼によりマウスを生け贄に捧げます。解剖トレイにマウスを置き、70％エタノールで滅菌します。半ば腹部に切開を行い、下肢をカバーする皮膚を含むマウスの先端部から皮膚を除去。 </li><li>半無菌フード内でマウスを解剖。大腿骨と脛骨を解除するには、膝関節以下上記の大腿骨と脛骨をクリップ。はさみを使用して、骨から筋肉を外し、完全RPMI 5ミリリットルを含む6センチペトリ皿に骨を置きます。 </li><li>無菌培養フードに移動します。完全RPMIで27G針に接続された3ミリリットル注射器を埋めます。はさみを使用して、骨の両端を切断し、静かにそれぞれの端から一度完全RPMIを注入することによって、骨の外に骨髄をフラッシュするために27Gの針を挿入します。 </li><li>細胞を上下に3-5回、針なし注射器を用いて培養皿で穏やかにピペッティングにより単一細胞懸濁液を準備します。 </li><li> 500×gで5分間、細胞を遠心分離し、上清をデカント。 </li><li> 1ミリリットルACKバッファー（150mMのNH 4 Clを 、10 mMのKHCO 3、0.1mMのEDTA）で1分間インキュベートし、完全RPMIの10容量で希釈して反応を停止で溶解赤血球。細胞が重力によって死んだ細胞塊および組織破片をダウンペレット化するために、5分間放置します。 注意：これは原因で生細胞の沈降に細胞の損失になりますように5分以上を立たないでください。 </li><li>ゆっくりと破片を残して新しいチューブに細胞含有上清をデカント元のチューブ内の背後にあります。 </li><li>その後、上清を除去し、廃棄し、細胞をペレットに500×gで5分間遠心します。 </li><li> 100μlのFACS緩衝液（1×PBS + 2％FBS + 1mMのEDTA）中（通常は4-6×10 7のBM細胞は、1つのマウスから得られる）総BM細胞を再懸濁します。 </li></ol> 3のCLPのFACS分析およびソート<ol><li> Fc受容体をブロックするために、1〜2分間抗CD16 / 32（ハイブリドーマ上清クローン2.4G2、50μL/反応または1-2μgの/反応）、FACS緩衝液中の細胞への（ステップ2.9）を追加します。 注：抗CD16 / 32はまた、顆粒球、単球およびBリンパ球などのFc受容体を発現する細胞に対する抗体の非特異的結合を防止するために添加されたFcブロックと呼ば​​れます。 </li><li>同時に周囲光を回避し、氷上で15分間（4×10 7細胞）以下の蛍光色素標識抗体で細胞を染色します。抗体：抗CD3を含むPE結合系統マーカー（0.2μgの各）（17A2）、抗CD8（53-6.7）、抗B220（RA3-6B2）、抗CD19（eBio1D3）、抗CD11b（M1 / 70）、抗GR-1（RB6-8C5）、抗Thy1.1（HIS51）、抗NK1.1（PK136）、抗TER119（TER-119）、および抗MHC-II（NIMR-4）、0.2μgの抗c-Kitの-PerCPを-のCy5.5 （2B8）、1μgの抗SCA-1-FITC（D7）、0.4μgの抗M-CSFR-APC（AFS98）、および0.2μgの抗IL-7Rα-PE-Cy7の（A7R34）。 </li><li> 3ミリリットルFACS緩衝液で細胞を洗浄し、500×gで5分間遠心します。 </li><li> 40μMのセルストレーナーを通して300μlのFACS緩衝液およびフィルタ細胞で細胞を​​再懸濁します。 </li><li>残りの細胞を回収し、同じセルストレーナーを使用してチューブをソートする無菌FACSにフィルタリングするために、追加の100μlのFACS緩衝液でチューブを洗浄します。 </li><li>信号検出のための適切なフィルタおよび電圧を使用して染色した後すぐにフローサイトメトリー分析を行います。 PE-およびPE-Cy7の-labelded抗体および633nmのラスを検出するために561 nmレーザー、FITC-、PerCPを-のCy5.5標識抗体を検出するために488nmのレーザーを使用してERは、APC標識抗体を検出します。 </li><li>セルソーターを用いて、IL-7Rα+（ステップ3.2で染色されたように） -のc-kit は int SCA-1 INT ​​M-CSFR -以下のマーカーLINに従ってソートアウトのCLP。クッションとして完全RPMI 8 mlを含む15mlチューブで選別された細胞を収集します。 </li><li>実行終了時にセルソーターによって示されるように選別された細胞の絶対数を記録します。一般的に、5×10 4のCLPは1マウスのBMから取得することができます。 </li></ol> 4.共培養のCLPとAC-6 <ol><li> 、500×gで10分間、ステップ3.7からの選別された細胞を遠心分離し、上清を除去し、5×10 4細胞/ mlの周りに細胞密度を得るために十分な完全RPMIで細胞ペレットを再懸濁します。シード500細胞/ウェルのステップ1.4で調製したフィーダー細胞を含む12ウェルプレートに。 </li><li> 100 ngの追加/ mlのFL 19（HU-のFlt3L-IgはM.マンツ、Universiによって提供される発現系を使用して、社内で発生しましたTY病院チューリッヒ、スイス）と37°Cの 、顕微鏡下でのDC開発の定期的な視覚的な監視と5％CO 2でインキュベートします。 注：市販の組換えヒトまたはマウスFL FLは、DCの発達をサポートするHU-のFlt3L-Igのための代替物として使用することができます。 </li><li> 12日目に上清中の細胞を収集し、得​​られた上清を組み合わせた0.5ミリリットル完全RPMI培地で1回、ウェルを洗浄します。 0.5ミリリットルに新鮮な培地を追加し、セルスクレーパーで接着細胞をスクラップ。 </li><li> 500×gで5分間の部品や遠心機の両方からの細胞含有培地を組み合わせます。 </li><li> 50μlのFACS緩衝液中で上清をデカントし再懸濁した細胞。 50μlの抗CD16 / 32ハイブリドーマ上清を加え、Fc受容体をブロックするために1~2分間インキュベートします。 </li><li>列挙し、次抗体（0.05μgの各）抗CD11cの-APC（N418）、抗CD11b-FITCおよび抗B220-PEで、すべての細胞を染色。 </li><li>洗って説明するように、細胞を遠心分離ステップ3.3でD。 </li><li> 、100μlのFACS緩衝液中で細胞を再懸濁したCD11c +の上のゲートとのCDC（のCD11c +のCD11b + B220 - ）のために分析したpDC（CD11cの+のCD11b - B220 +）の17。 </li></ol>

<ol>
	<li>Hartwig, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fcgamma+receptor-mediated+antigen+uptake+by+lung+DC+contributes+to+allergic+airway+hyper-responsiveness+and+inflammation.">Fcgamma receptor-mediated antigen uptake by lung DC contributes to allergic airway hyper-responsiveness and inflammation.</a> Eur. J. Immunol. 40, 1284-1295 (2010).</li><li>Merad, M., Sathe, P., Helft, J., Miller, J., Mortha, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dendritic+cell+lineage:+ontogeny+and+function+of+dendritic+cells+and+their+subsets+in+the+steady+state+and+the+inflamed+setting.">The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting.</a> Annu. Rev. Immunol. 31, 563-604 (2013).</li><li>Mildner, A., Jung, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Function+of+Dendritic+Cell+Subsets.">Development and Function of Dendritic Cell Subsets.</a> Immunity. 40, 642-656 (2014).</li><li>Swiecki, M., Colonna, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+functions+of+plasmacytoid+dendritic+cells+during+viral+infections,+autoimmunity,+and+tolerance.">Unraveling the functions of plasmacytoid dendritic cells during viral infections, autoimmunity, and tolerance.</a> Immunol. Rev. 234, 142-162 (2010).</li><li>Liu, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IPC:+professional+type+1+interferon-producing+cells+and+plasmacytoid+dendritic+cell+precursors.">IPC: professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors.</a> Annu. Rev. Immunol. 23, 275-306 (2005).</li><li>Merad, M., Manz, M. G. <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+homeostasis.">Dendritic cell homeostasis.</a> Blood. 113, 3418-3427 (2009).</li><li>Ghosh, H. S., Cisse, B., Bunin, A., Lewis, K. L., Reizis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+expression+of+the+transcription+factor+e2-2+maintains+the+cell+fate+of+mature+plasmacytoid+dendritic+cells.">Continuous expression of the transcription factor e2-2 maintains the cell fate of mature plasmacytoid dendritic cells.</a> Immunity. 33, 905-916 (2010).</li><li>Karsunky, H., Merad, M., Cozzio, A., Weissman, I. L., Manz, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flt3+ligand+regulates+dendritic+cell+development+from+Flt3++lymphoid+and+myeloid-committed+progenitors+to+Flt3++dendritic+cells+in+vivo.">Flt3 ligand regulates dendritic cell development from Flt3+ lymphoid and myeloid-committed progenitors to Flt3+ dendritic cells in vivo.</a> J. Exp. Med. 198, 305-313 (2003).</li><li>Manz, M. G., Traver, D., Miyamoto, T., Weissman, I. L., Akashi, K. <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+potentials+of+early+lymphoid+and+myeloid+progenitors.">Dendritic cell potentials of early lymphoid and myeloid progenitors.</a> Blood. 97, 3333-3341 (2001).</li><li>D'Amico, A., Wu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+early+progenitors+of+mouse+dendritic+cells+and+plasmacytoid+predendritic+cells+are+within+the+bone+marrow+hemopoietic+precursors+expressing+Flt3.">The early progenitors of mouse dendritic cells and plasmacytoid predendritic cells are within the bone marrow hemopoietic precursors expressing Flt3.</a> J. Exp. Med. 198, 293-303 (2003).</li><li>Sathe, P., Vremec, D., Wu, L., Corcoran, L., Shortman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+differentiation:+myeloid+and+lymphoid+pathways+to+murine+plasmacytoid+dendritic+cells.">Convergent differentiation: myeloid and lymphoid pathways to murine plasmacytoid dendritic cells.</a> Blood. 121, 11-19 (2013).</li><li>Shigematsu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmacytoid+dendritic+cells+activate+lymphoid-specific+genetic+programs+irrespective+of+their+cellular+origin.">Plasmacytoid dendritic cells activate lymphoid-specific genetic programs irrespective of their cellular origin.</a> Immunity. 21, 43-53 (2004).</li><li>Reizis, B., Bunin, A., Ghosh, H. S., Lewis, K. L., Sisirak, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmacytoid+dendritic+cells:+recent+progress+and+open+questions.">Plasmacytoid dendritic cells: recent progress and open questions.</a> Annu. Rev. Immunol. 29, 163-183 (2011).</li><li>Inaba, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+large+numbers+of+dendritic+cells+from+mouse+bone+marrow+cultures+supplemented+with+granulocyte/macrophage+colony-stimulating+factor.">Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.</a> J. Exp. Med. 176, 1693-1702 (1992).</li><li>Naik, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+plasmacytoid+and+conventional+dendritic+cell+subtypes+from+single+precursor+cells+derived+in+vitro+and+in+vivo.">Development of plasmacytoid and conventional dendritic cell subtypes from single precursor cells derived in vitro and in vivo.</a> Nat. Immunol. 8, 1217-1226 (2007).</li><li>Onai, N., et al. <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+clonogenic+common+Flt3+M-CSFR++plasmacytoid+and+conventional+dendritic+cell+progenitors+in+mouse+bone+marrow.">Identification of clonogenic common Flt3+M-CSFR+ plasmacytoid and conventional dendritic cell progenitors in mouse bone marrow.</a> Nat. Immunol. 8, 1207-1216 (2007).</li><li>Chen, Y. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+type+I+IFN-Flt3+ligand+axis+augments+plasmacytoid+dendritic+cell+development+from+common+lymphoid+progenitors.">A type I IFN-Flt3 ligand axis augments plasmacytoid dendritic cell development from common lymphoid progenitors.</a> J. Exp. Med. 210, 2515-2522 (2013).</li><li>Whitlock, C. A., Tidmarsh, G. F., Muller-Sieburg, C., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cell+lines+with+lymphopoietic+activity+express+high+levels+of+a+pre-B+neoplasia-associated+molecule.">Bone marrow stromal cell lines with lymphopoietic activity express high levels of a pre-B neoplasia-associated molecule.</a> Cell. 48, 1009-1021 (1987).</li><li>Onai, N., Obata-Onai, A., Tussiwand, R., Lanzavecchia, A., Manz, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+Flt3+signal+transduction+cascade+rescues+and+enhances+type+I+interferon-producing+and+dendritic+cell+development.">Activation of the Flt3 signal transduction cascade rescues and enhances type I interferon-producing and dendritic cell development.</a> J. Exp. Med. 203, 227-238 (2006).</li><li>Szilvassy, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukemia+inhibitory+factor+upregulates+cytokine+expression+by+a+murine+stromal+cell+line+enabling+the+maintenance+of+highly+enriched+competitive+repopulating+stem+cells.">Leukemia inhibitory factor upregulates cytokine expression by a murine stromal cell line enabling the maintenance of highly enriched competitive repopulating stem cells.</a> Blood. 87, 4618-4628 (1996).</li><li>Arcanjo, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+of+heparan+sulfate+derived+from+murine+hemopoietic+stromal+cell+lines:+a+bone+marrow-derived+cell+line+S17+and+a+fetal+liver-derived+cell+line+AFT024.">Biochemical characterization of heparan sulfate derived from murine hemopoietic stromal cell lines: a bone marrow-derived cell line S17 and a fetal liver-derived cell line AFT024.</a> J. Cell. Biochem. 87, 160-172 (2002).</li><li>Onai, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clonogenic+progenitor+with+prominent+plasmacytoid+dendritic+cell+developmental+potential.">A clonogenic progenitor with prominent plasmacytoid dendritic cell developmental potential.</a> Immunity. 38, 943-957 (2013).</li><li>Whitlock, C. A., Muller-Sieburg, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+B-lymphoid+cultures+from+murine+bone+marrow+establishment+and+cloning+by+using+stromal+cell+line+AC+6.21.">Long-term B-lymphoid cultures from murine bone marrow establishment and cloning by using stromal cell line AC 6.21.</a> Methods Mol. Biol. 75, 231-248 (1997).</li><li>Chen, Y. -. L., Chang, S., Chen, T. -. T., Lee, C. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Generation+of+Plasmacytoid+Dendritic+Cell+from+Common+Lymphoid+Progenitors+by+Flt3+Ligand.">Efficient Generation of Plasmacytoid Dendritic Cell from Common Lymphoid Progenitors by Flt3 Ligand.</a> PLoS ONE. 10 (8), e0135217 (2015).</li></ol>

Authors have nothing to disclose.

ここでは、のCLPの少数から、特にDCの堅牢な世代のためのin vitro培養系を説明し、pDCに。この培養系の独自性は、フィーダーとしてのAC-6細胞、ストローマ細胞株の使用によるものです。このアプローチは、IL-7、SCF、M-CSFおよびFL 20などのサイトカインが、また、DCの発達をサポートするのに必要な細胞-細胞接触21だけでなく、提供することが示されています。 AC-6細胞は?...

樹状細胞（DC）は、1免疫応答の制御に重要な役割を果たしているプロフェッショナル抗原提示細胞です。 DCは不均一であるが、それらは、大きく2つの集団に分類することができ、従来のDC（のCDC）および形質のDC（pDCに）2,3。リンパ器官に加えて、のCDCとのpDCは、肺、腸および皮膚2を含む組織中に見出されます。 CDCとのpDCの形態はのCDCは、デンドライト状の突起と以上のプラズマ細胞様であることのpDCの形状を呈すると、異なります。また、一般的なマウスDCマーカーCD11cのは、より高度のpDC上でのCDCより上で発現されます。 pDCに2が行うよりも、MHCクラスIIの高いレベルと共刺激分子を発現どちらのCD24 +のCDC、 -のCDCとのCD11b -また、のCDCは、さらにのCD11b + CD24に分けることができます。成熟のpDCは、一方で、選択シグレックHおよびBST2 4を発現することが示されています。 Functionally、のCDCは、pDCにはよりも優れた抗原提示細胞です。しかし、pDCには、私は、ウイルス感染または炎症性刺激5時に型インターフェロンの膨大な量を生成することができます。 両方のCDCとのpDCは短命であるため、常に骨髄（BM）6,7内の前駆細胞から補給する必要があります。致死的に照射したマウスに共通の骨髄前駆細胞（のCMP）とリンパ球系共通前駆細胞（のCLP）の養子移入はのCDCとのpDCは、両方の系統8-10から生成することができることを実証しています。しかし、RAG1 / 2を発現し、IgH遺伝子座11,12に再配置DJセグメントを所有するpDCのサブセットがあります。これらの細胞はまた、B220マーカーの発現は、核酸感知受容体（TLR7 / TLR9）、および転写因子（SPIBとBcl11a）13を含む Bリンパ球との分子の類似性を共有すると、すべてがリンパ系列の署名であると考えられています。したがって、CLPsがあるため、系統の類似のpDCのインビトロ生成のための良い選択かもしれません。 ヒトおよびマウスの両方でのCDCとのpDCの頻度が非常に低いが6、DCは、特にたCDCは、（例えば、GM-CSF 11,14またはFlt3リガンドなどのサイトカインの存在下で、BMまたは前駆細胞からインビトロで生成することができるFL ）フィーダーフリーシステム11,15,16を使用 。しかし、残念ながら、それは、FL 11,15,16 を用いてインビトロでのpDCの多くを生成することは不可能です。以前に我々は、pDCには、効率的にAC-6フィーダシステム17を使用してのCLP からインビトロで生成することができることを実証しました。培養系においてAC-6間質細胞株を使用することの利点は、それが細胞 - 細胞接触とのCLPからのpDCの多数の生成をサポートするサイトカインの分泌を提供することです。このシステムの生産は非常に堅牢ですが、下記の手順を慎重にレプリケーションが私を必要とされますn個の順序のpDCの効率的な生成を確実にします。

<table>
	<tbody>
		<tr>
			<td>Anti-mouse Ly6g/Ly6c (PE), clone RB6-8C5</td>
			<td>Biolegend</td>
			<td>108408</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse NK1.1 (PE), clone PK136</td>
			<td>Biolegend</td>
			<td>108708</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse&#160; CD11b (PE), cloneM1/70</td>
			<td>Biolegend</td>
			<td>101208</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse CD19 (PE), clone eBio1D3</td>
			<td>Biolegend</td>
			<td>115508</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse B220 (PE), clone RA3-6B2</td>
			<td>Biolegend</td>
			<td>103208</td>
			<td>linage marker/FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse CD3 (PE), clone 17A2</td>
			<td>Biolegend</td>
			<td>100308</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse CD8a (PE), clone 53-6.7</td>
			<td>Biolegend</td>
			<td>100707</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse MHC-II (PE), clone NIMR-4</td>
			<td>Biolegend</td>
			<td>107608</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse Ter119 (PE), clone TER-119</td>
			<td>Biolegend</td>
			<td>116208</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse Thy1.1 (PE), clone HIS51</td>
			<td>eBioscience</td>
			<td>12-0900-83</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse M-CSFR (APC), clone AFS98</td>
			<td>Biolegend</td>
			<td>135510</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse c-Kit (PerCP/Cy5.5), clone 2B8</td>
			<td>Biolegend</td>
			<td>105824</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse Sca-1 (FITC), clone D7</td>
			<td>Biolegend</td>
			<td>108106</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse IL-7Ra (PE/Cy7), clone A7R34</td>
			<td>Biolegend</td>
			<td>135014</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11c (PerCP/Cy5.5), clone N418</td>
			<td>Biolegend</td>
			<td>117328</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b (FITC), clone M1/70</td>
			<td>Biolegend</td>
			<td>101206</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>FACSAria III</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Cell sorter</td>
		</tr>
		<tr>
			<td>FACS sorting tube&#160;</td>
			<td>BD Biosciences</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>FlowJo LLC</td>
			<td></td>
			<td>Flow analysis sofrware</td>
		</tr>
	</tbody>
</table>

in vitro generation of murine plasmacytoid dendritic cells from common lymphoid progenitors using the ac-6 feeder system

形質細胞様樹状細胞（pDC）感染に応答して、または炎症応答の間に活性化された細胞を産生Iインターフェロン強力型（IFN-I）です。残念ながら、pDCの機能の研究は、リンパ器官で、その低周波によって妨げられ、in vitroでのDC生成のための既存の方法は、主にpDCを超えるのCDCの生産を好みます。ここでは、効率的にin vitroでのリンパ球系共通前駆細胞（のCLP）からのpDCを生成するためのユニークなアプローチを提示します。具体的には、プロトコルは、骨髄からのCLPを浄化し、Flt3リガンドの存在下でγ線を照射し、AC-6フィーダー細胞との共培養により、pDCにを生成する方法の詳細を説明しました。この培養系のユニークな特徴は、のCLPは、AC-6細胞の下に移動して、石畳の領域形成細胞になることのpDCを拡大するための重要なステップです。形態学的に異なるDCは、すなわちたpDCとのCDCは、7の組成物で約2週間後に生成されました最適な条件の下で0から90パーセントのpDCに。通常、この方法によって生成されたpDCの数はおおよそのCLPの数の100倍がシードされます。したがって、これは、確実に、これらの細胞の発達と機能にさらなる研究を容易にするために必要なのpDCを大量に生成すると、新規システムです。

4-6x10 7 BM細胞の総数は、典型的には、大腿骨一6~8週齢の野生型C57BL / 6マウスの脛骨から単離されます。 CLPを整理するには、総BM細胞は、系統マーカー（CD3、CD8、B220、CD19、CD11bの、GR-1、Thy1.1、NK1.1、TER119、およびMHC-II）に対するPE結合抗体で染色され、抗-c-Kitの-PerCPを/ Cy5.5を、抗SCA-1-FITC、抗M-CSFR-APCおよび抗IL-7Rα-PE / Cy7の、分析し、セルソーターでソート。 CLPのための選別戦略は<stron...

Watch this Scientific Journal Video about In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System at JoVE.com

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

AC-6フィーダシステムを使用してリンパ球系共通前駆細胞からマウス形質細胞様樹状細胞のインビトロ生成に

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

in-vitro-generation-murine-plasmacytoid-dendritic-cells-from-common

Research

JoVE Journal

Immunology and Infection

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

8.3K ビュー.  National Taiwan University College of Medicine. In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand. 

ビデオ: In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

追跡調査のためのQdot ®二ナノとマウス骨髄由来樹状細胞の世代とラベリング

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

免疫・感染学

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer&#039;s Patches

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

マウスのパイエル板のリンパ球や樹状細胞を単離し、免疫染色

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

黒色腫と扁平上癌幹細胞を用いた新規樹状細胞ワクチンの生成

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

造血前駆細胞の養子移入を使用して、パイエル板におけるマウス形質細胞様樹状細胞の開発を評価

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1感染CD4の生得センシングを評価<sup&gt; +</sup&gt;使用して形質細胞様樹状細胞によるT細胞<em&gt;エクスビボ</em&gt;共培養系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

代謝表現型が異なる未熟、熟女と寛容原性樹状細胞の生成

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

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. ...

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 ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

生体外でマウスのナチュラル キラー細胞の大量生産のため新規無料の送り装置システム

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...