著者らは、マウスの皮膚におけるスポロゾイトの運動性のin vivoイメージングに教えるためのパトリシアBaldacci、ヴァネッサLagalとザビーネThiberge重要な読書のために、イリーナDobrescuとザビーネThiberge写真を撮るのヘルプおよびポーリンFormaglioに感謝します。また、蚊の飼育のための生産とハマダラカ（CEPIA-パスツール研究所）の感染のためにマレックSzatanikとセンターに感謝したいと思います。この研究は、ラボラトリードール・エクセレンス &quot;新興感染症の統合生物学」（無許可。ANR-10-LABX-62-IBEID）からAX​​Aリサーチファンドと資金によってサポートされていました。

<html>すべての手順は、パスツール研究所の委員会や地元動物実験に関する倫理委員会（倫理委員IDF-パリ1、パリ、フランス、契約番号：2012から0015）によって承認された際は適用される基準および規則に従って行いました。 1.材料および試薬<ol><li>以前21記載されているように3〜5日出現とリアの後に感染したマウスを餌に女性ハマダラカ蚊（Sda500株）を使用します。 </li><li>構成的熱ショックタンパク質70（HSP70）プロモーターの制御下で緑色蛍光タンパク質（GFP）遺伝子を発現する寄生虫原虫ベルゲイ ANKAクローンを使用します。これは、寄生虫のライフサイクル22を通して明るい蛍光が得られます。 </li><li>雌のC57BL / 6JRjマウス（7週齢）を使用します。 注：記載されているプロトコルで使用されるすべての試薬 ​​は、 マテリアル/機器のできる Tに記載されています。 </li></ol>_title &quot;&gt; 2。蚊唾液腺からの放射弱毒化スポロゾイトの単離<ol><li>感染血液食事の後18-25日の間、収集し、以前に21記載されているように氷上で15 mlのチューブに蚊をコールド麻酔。慎重にチューブを反転させてペトリ皿に移します。氷の上で昆虫を維持し、γ-又はX線照射の12 kradの線量に蚊を公開します。 </li><li>以前に21を説明したように、照射蚊の唾液腺からの弱毒化スポロゾイトを分離します。氷上で1×ダルベッコのリン酸緩衝生理食塩水（DPBS）の（15μL程度）小音量で感染した唾液腺を収集し、静かにスポロゾイトを解放するためにそれらをつぶします。 注：彼らの強い発現によって証明されるように重要では小容量で高濃縮寄生虫懸濁液の注入は、事前に選択され、十分に感染した蚊の数が多いから、唾液腺の十分な数を収穫することが不可欠です緑色蛍光。 </li><li>塊と蚊の破片を除去するために、低接着1.5ミリリットルマイクロ遠心チューブに適合した35μmのセルストレーナーキャップを介してスポロゾイトサスペンションをフィルタリングします。 </li><li>以前に21を説明したように、単離されたスポロゾイトの合計数をカウントし、83,000スポロゾイト/μLを得るために、冷1×DPBSで寄生虫懸濁液の濃度を調整します。次のステップを継続しながら氷上で寄生虫を保管してください。 </li><li>ネガティブコントロールとして使用される非感染蚊から唾液腺の同等の数を収集し、手順2.2と2.3で説明したようにサンプルを処理します。懸濁液中の唾液腺抽出物の類似の濃度を得るために、上記のように試料を希釈します。 注：スポロゾイトは、2時間の最大氷上で保存することができます。しかし、理想的にはそれらは唾液腺を粉砕後1時間以内に注入されています。 </li></ol>皮膚ラへのスポロゾイトの3インジェクション耳のヤー<ol><li>ケタミンの腹腔内注射によって動物を麻酔（50mg / kgの）/キシラジン（5mg / kg）の混合物。マウスは無意識の状態であることと、角膜の乾燥を防ぐために、眼に眼軟膏を適用するために5-8分待ちます。両方のリアの足に優しいつま先のピンチを実行することによって、麻酔の深さを評価します。 注：麻酔の投与量は、20分未満継続するので、次のステップが迅速に行わなければなりません。 </li><li>以前に実体顕微鏡（ 図1A）下に置かれた明確なテープで腹側を固定することにより、マウスの耳介を安定させます。優しく血管系の損傷を避けるために、耳を押します。 </li><li>再懸 ​​濁した寄生虫10の0.6μlの10μlのシリンジ/ 35ゲージの面取り針をロードします。 </li><li>表皮の下に、注意深く耳（ 図1A）の背側に針を挿入することにより、4注射部位（部位あたり0.15μL）でスポロゾイトサスペンションを提供し、とベベルアップ、任意の血管を避けるように注意しながら。針は、それを削除する前に、注入物質の逆流を防止するために数秒のために真皮内に残ることを許可します。手順（ 図1Bおよび1C）の最後に各注射部位の特性丘疹を観察します。 </li><li>注入後、慎重にテープから耳を削除します。 </li><li>手順3.2から3.4に従うことによって寄生虫の同用量と反対側の耳に注入します。 </li><li>単独の注射と真皮における蚊材料の堆積によって媒介される炎症反応を評価するために、1×DPBSの0.6μlあるいは非感染蚊から1×DPBS +唾液腺抽出物0.6μlのいずれかでの2つの追加のマウスを注入します。 </li><li>ケージに戻ってそれを置く前に、麻酔からのマウスの回復を監視します。 注：皮膚における適切なスポロゾイト堆積は、蛍光顕微鏡検査（ 図2Aおよび図2Bによってモニターすることができますprevisously 23記載されているように）、マウスを用意しています。 </li></ol> 4.皮膚及び流入領域リンパ郭清<ol><li>ダルベッコ改変イーグル培地（DMEM）+ 25 mMの400 U / mlのコラゲナーゼおよび50μg/ mlのデオキシリボヌクレアーゼ（DNアーゼ）を補充した4-（2-ヒドロキシエチル）-1-ピペラジンエタンスルホン酸（HEPES）、次のように標準培地（SM）を調製。 </li><li> DLNサンプル用ウェル当たり4.5ミリリットルSM +70μmのセルストレーナー（プレートA）および皮膚サンプル用ウェル当たり4.5ミリリットルSM（プレートB）と氷上で2 6ウェル平底組織培養プレートを準備します。 </li><li>スポロゾイト接種後の所望の時点では、ケタミン（125mgの/キログラム）/キシラジン（12.5ミリグラム/キログラム）混合前に頸椎脱臼の腹腔内注射によってマウスを麻酔。マウスは臨床死の兆候を示しているとすぐに解剖を開始していることを確認します。 注：骨髄細胞動員の動態は、INJ後の異なる時点で検査することができますection（以前に14を説明するように、すなわち 2時間、4時間および24時間）。 </li><li>汚染の可能性を減少させるために70％エタノールを接種し、耳と対応するネック領域を消毒します。 </li><li>滅菌はさみやピンセットを用いて、無菌的に周囲の脂肪を収集せずに、近位耳のDLNを分析。 jugulo-carotidian溝の最初の切開を行い、手術現場から毛を取り除きます。周囲の組織に比べて灰色がかった表示されますDLNを露出させるために2鉗子で切開を伸ばします。 </li><li>その除去を容易にするために鉗子でDLNの上に慎重に結合組織を挟みます。リンパ組織（ 図3Aおよび3B）の下に鉗子を置くことによってDLNを抽出します。よく含む4.5ミリリットルSM +70μmのセルストレーナー（プレートA）にDLNを置きます。 </li><li>無菌鋭いハサミやピンセット（ 図4A）を使用して、全体の接種耳を収穫。 SEPARピンチと2鉗子（ - 4E 図4B）で終わるカットを離間さによって耳の背側と腹側の側面を食べました。両方の組織は完全培地中に浸漬されていることを確認することも含む4.5ミリリットルSM（プレートB）にそれらを配置します。 注：実験の信頼性を向上させ、関心対象の細胞の数を増加させるために、プール2は、サンプルあたり同じマウスからの耳又は2 dLNs注入しました。 </li><li>耳と1×DPBSまたは唾液腺抽出物のいずれかを接種したマウスのdLNs並列に収穫し、処理。負の単一の染色補正コントロールのナイーブマウスから採取した2つの追加の耳とリンパ節サンプルを含みます。 </li></ol>リンパ節から5細胞の単離<ol><li>穏やかな攪拌下で15分間、37℃+ 5％CO 2でのリンパ節（プレートA）にインキュベートします。コラゲナーゼおよびDNアーゼACTIを中和するために、ウェルあたり10 mMの最終エチレンジアミン四酢酸（EDTA）を追加vities。氷の上で次の手順を実行します。 </li><li> 5mLのシリンジプランジャーの先端を用いてリンパ節を解離します。残っているすべてのまでストレーナーに対して円運動でEnterキーを押して、白の結合組織です。 </li><li> 500μlの1×DPBS + 2％ウシ胎児血清（FBS）+ 2mMのEDTAで二回セルストレーナーを洗浄します。 15ミリリットルチューブにウェルのボリューム全体を転送します。 </li><li> 500μlの1×DPBS + 2％FBS + 2mMのEDTAでも二回を洗浄し、15ミリリットルチューブにボリュームを移します。氷の上にチューブを保管してください。 </li></ol>耳の皮膚から6細胞の単離<ol><li>穏やかな攪拌下で1時間、37℃+ 5％CO 2で耳のリーフレット（プレートB）をインキュベートします。 注：インキュベーションの20分後、組織消化を促進し、残りの40分間バックインキュベーター内のサンプルを入れて小片にハサミで耳のリーフレットを切りました。単一細胞はインキュベーション時間の終了時に培地中に観察することができます。 </li><li> 10mMの最終を追加<html>ウェルあたりEDTA、コラゲナーゼおよびDNアーゼ活性を中和します。氷の上で次の手順を実行します。 </li><li>耳組織断片とよく1ミリリットルピペットを用いて、70μmのセルストレーナーを含む新しい培地中の全容量を転送します。より簡単に耳組織断片を収集するためにチップの先端から2〜3ミリメートルをカット。 </li><li> 5ミリリットルの注射器プランジャの先端を使用して、耳の組織断片を解離します。残っているすべてのまでストレーナーに対して円運動でEnterキーを押して、白の結合組織です。 </li><li> 500μlの1×DPBS + 2％FBS + 2mMのEDTAで二回セルストレーナーを洗浄します。 </li><li> 70μmのセルストレーナーを介して50ミリリットルチューブに入ってものボリューム全体を転送します。 </li><li> 500μlの1×DPBS + 2％FBS + 2mMのEDTAとをよくすすぎ、50ミリリットルチューブにボリュームを移します。チューブを氷上に保管してください。 注：皮膚およびDLN組織から全細胞を分離する別の手順を図5にまとめます。 </li></ol>タイトル &quot;&gt; 7。数及び採取した細胞の生存率<ol><li> 4℃で450×gで5分間、皮膚とDLNサンプルを遠心分離し、上清を捨てます。静かに300μlの1×DPBS + 2％FBS + 2mMのEDTAでペレットを再懸濁。 </li><li>血球計数器を用いて皮膚及びDLNから単離された細胞の総数をカウントするために、各試料の少量を収集します。細胞生存率を決定するために、0.04％トリパンブルーを追加します。 </li></ol>非8.ブロッキング抗原特異的結合<ol><li>非標識CD16 / CD32のFc-ブロック抗体10μg/ mlを追加します。 （長く行くことができます）、4℃で20分間インキュベートします。 </li><li>冷たい1×DPBS + 2％FBS + 2mMのEDTAの2ミリリットルを追加します。 4℃で450×gで5分間サンプルを遠心し、上清を捨てます。静かに+ 2％FBS + 2mMのEDTA300μlの1×DPBSでペレットを再懸濁し、氷上でサンプルを維持します。 </li></ol> 9.染色皮膚及びリンパ節骨髄細胞<ol><li>以前にブロックされたスキーをインキュベートn及びDLNライブ/デッド・トラッカー（ すなわち 4 &#39;、6-ジアミジノ-2-フェニルインドールまたはDAPI）、抗CD45および抗CD11b特異的抗体で細胞を単離しました。 注意：利息の骨髄細胞亜集団、特に希少なものを豊かにするためには、CD45 +細胞およびリンパ球は、それぞれ皮膚から精製することができるか、細胞選別を容易にする磁気ビーズによってDLNから枯渇。 DLNから単離された細胞のほとんどがCD45 +であるので、抗CD45の使用は必須ではありません。他のマーカーは、正または負のゲーティング戦略により対象の骨髄細胞の亜集団を同定するために含めることができます。このプロトコルでは、DLNに動員骨髄細胞を、CD8α+細胞（全T細胞コンパートメントは、抗CD3特異的抗体を用いて標的とすることができる）を除くことによって濃縮しました。 </li><li>暗所で4℃で30分間インキュベートします。 500μlの1×DPBS + 2％FBS + 2mMのEDTA、遠心分離機4℃で450×gでサンプル5分を追加し、supeを捨てますrnatant。二回洗浄ステップを繰り返します。 </li><li> 300μlの1×DPBS + 2％FBS + 2mMのEDTAを含む試料を再懸濁します。 35μmのセルストレーナーを通してFACSチューブにボリューム全体を転送します。 100μlの1×DPBS + 2％FBS + 2mMのEDTAでフィルターを洗浄します。 </li><li>フローサイトメーターで染色した細胞を実行します。死細胞とダブレットを排除する-ゲートは、単一細胞（FSC-W DAPI）を生きます。プロットCD45 +のCD11b +皮膚（ 図6A）のためのイベントと（CD45 +）CD8α - DLN（ 図6B）のためのCD11b +イベント。 </li></ol>

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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=Lymph-node+resident+CD8&#945;++dendritic+cells+capture+antigens+from+migratory+malaria+sporozoites+and+induce+CD8++T+cell+responses.">Lymph-node resident CD8&#945;+ dendritic cells capture antigens from migratory malaria sporozoites and induce CD8+ T cell responses.</a> PLoS Pathog. 11 (2), e1004637 (2015).</li><li>da Silva, H. B., 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=Early+skin+immunological+disturbance+after+Plasmodium-infected+mosquito+bites.">Early skin immunological disturbance after Plasmodium-infected mosquito bites.</a> Cell Immunol. 277 (1-2), 22-32 (2012).</li><li>Mac-Daniel, 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=Local+immune+response+to+injection+of+Plasmodium+sporozoites+into+the+skin.">Local immune response to injection of Plasmodium sporozoites into the skin.</a> J Immunol. 193 (3), 1246-1257 (2014).</li><li>Ribeiro-Gomes, F. L., Peters, N. C., Debrabant, A., Sacks, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+capture+of+infected+neutrophils+by+dendritic+cells+in+the+skin+inhibits+the+early+anti-leishmania+response.">Efficient capture of infected neutrophils by dendritic cells in the skin inhibits the early anti-leishmania response.</a> PLoS Pathog. 8 (2), e1002536 (2012).</li><li>Thiberge, S., 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=In+vivo.+imaging+of+malaria+parasites+in+the+murine+liver.">In vivo. imaging of malaria parasites in the murine liver.</a> Nat Protoc. 2 (7), 1811-1818 (2007).</li><li>Ishino, T., Orito, Y., Chinzei, Y., Yuda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+calcium-dependent+protein+kinase+regulates+Plasmodium+ookinete+access+to+the+midgut+epithelial+cell.">A calcium-dependent protein kinase regulates Plasmodium ookinete access to the midgut epithelial cell.</a> Mol Microbiol. 59 (4), 1175-1184 (2006).</li><li>Amino, R., 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=Imaging+malaria+sporozoites+in+the+dermis+of+the+mammalian+host.">Imaging malaria sporozoites in the dermis of the mammalian host.</a> Nat Protoc. 2 (7), 1705-1712 (2007).</li><li>Ploemen, I. 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=Plasmodium+liver+load+following+parenteral+sporozoite+administration+in+rodents.">Plasmodium liver load following parenteral sporozoite administration in rodents.</a> Vaccine. 31 (34), 3410-3416 (2013).</li><li>Epstein, J. E., 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=Live+attenuated+malaria+vaccine+designed+to+protect+through+hepatic+CD8+T+cell+immunity.">Live attenuated malaria vaccine designed to protect through hepatic CD8 T cell immunity.</a> Science. 334 (6055), 475-480 (2011).</li><li>Roestenberg, M., 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=Long-term+protection+against+malaria+after+experimental+sporozoite+inoculation:+an+open-label+follow-up+study.">Long-term protection against malaria after experimental sporozoite inoculation: an open-label follow-up study.</a> Lancet. 377 (9779), 1770-1776 (2011).</li><li>Seder, R. A., 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=Protection+against+malaria+by+intravenous+immunization+with+a+nonreplicating+sporozoite+vaccine.">Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine.</a> Science. 341 (6152), 1359-1365 (2013).</li><li>Douradinha, B., 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=Genetically+attenuated+P36p-deficient+Plasmodium+berghei+sporozoites+confer+long-lasting+and+partial+cross-species+protection.">Genetically attenuated P36p-deficient Plasmodium berghei sporozoites confer long-lasting and partial cross-species protection.</a> Int J Parasitol. 37 (13), 1511-1519 (2007).</li><li>Geem, D., Medina-Contreras, O., Kim, W., Huang, C. S., Denning, T. 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著者は、彼らが競合する金融利害関係を持たないことを宣言します。

全スポロゾイトマラリアワクチンを用いたヒトの大規模なワクチン接種の観点では、克服すべき主な課題の一つは、成功した免疫化および保護24,25を確実にするために最適化された経路および寄生虫投与方法を開発することです。ヒトでは、生きた弱毒寄生虫（LAP）によって媒介される防御効果の評価は、自然蚊に刺さ2と同様に、皮内、皮下25,26およびIV免疫化の2...

マラリアは年間50万人以上を殺害し、世界最悪の感染症の一つです。 マラリア原虫 、疾患の原因物質による感染は、前赤血球（PE）相によって開始されます。このフェーズでは、雌ハマダラカ蚊によって宿主の皮膚に注入されたスポロゾイトは、血流を介して肝臓に到達し、赤血球に感染し、疾患の症状を引き起こす寄生形態に内部に肝細胞を区別する。 マラリア原虫のPE段は、抗マラリアワクチン接種のための特権標的を表します。実際、このような放射線弱毒化スポロゾイト（RAS）などのこれらのステージに対する弱毒化ワクチンを、生きて、遺伝的に逮捕された寄生虫（GAP）または化学的予防及びスポロゾイト（CPS）は、げっ歯類とヒト宿主1-9の両方を保護する能力を実証しています。齧歯類モデルでは、ほとんどのワクチン接種研究は、静脈内免疫を利用して行われます、防御効力の点でゴールドスタンダードです。しかし、皮膚のステージの説明と保護を誘発するのに皮膚関連の流入領域リンパ節（DLN）の重要性は、PE相の私達の認識を変え、注入の皮内経路の重要性を強調しています。 P.の生体内イメージングげっ歯類の皮膚に注入ベルゲイのスポロゾイトは、接種物のみ〜25％が血流を介して肝臓に到達したことを示しています。残りの〜75％は、近位DLN（〜15％）と少量変換し、皮膚細胞12,13の内側週間生きている残ることができる皮膚（〜50％）10,11、の間で分配します。また、その後の研究は、皮内免疫化後の効果的な防御免疫の確立は、主に寄生虫特異的CD8 + T細胞が14,15脾臓または肝臓-dLNsにわずかしか活性化され、皮膚DLNに起こることを説明しました。 同時にほとんどの研究は、はるかに少ない皮膚に注入生きた弱毒寄生虫、先天性免疫系との特にそれらの相互作用の運命について知られている、防御免疫応答の確立に関与するエフェクター細胞の特徴付けに集中しています。具体的には、CD8 + T細胞への寄生虫抗原取り込み、プロセシングおよび提示に関与する抗原提示細胞の特徴付けは、PE抗原捕捉は、皮膚及びDLNコンパートメントの両方において発生する可能性があることを知って、非常に重要です。前の生体内イメージング研究は、スポロゾイトと樹状細胞との間の初期の相互作用がDLN 10,17で観察された感染蚊に刺され16次肌に明るい蛍光のLys-GFP陽性細胞の早期流入を説明しました。より最近では、蚊によって皮膚に接種スポロゾイトは、皮膚の両方の樹状細胞および制御性T細胞の運動性を増大させることが報告されていますマウスを、抗原提示細胞の減少数は、DLN 18で観察されました。 我々は、白血球の皮膚とそれに対応するDLNで募集サブセットだけでなく、RAS 19の用量を免疫の皮内注射後の寄生虫と相互作用するものを同定し、より正確に定量化することを目的としました。この文脈において、我々は両方の組織から骨髄細胞（CD45 +のCD11b +）を単離し、マルチ=パラメトリックフローサイトメトリーによって関心の亜集団を特徴とします。 リーシュマニアの主要な皮膚感染20の初期の段階で説明した免疫応答と一致して、注射をスポロゾイトする主な宿主応答は、炎症性単球（CD45 +のCD11b続い多形核好中球（CD45 +のCD11b + Ly6G + Ly6C int型 ）の連続した募集で構成されてい+ Ly6G - differentiに基づいて識別されているLy6C +）Ly6GおよびLy6C表面マーカーのアル表現。 ここでは、感染した蚊の唾液腺から抽出されたRASの用量を免疫の皮内注射後のマウスの皮膚とDLNから骨髄細胞を単離するためのプロトコルについて説明します。再現性の皮内注射し、組織処理は、感染組織内の細胞集団を浸潤の表現型の変化を定量化するための重要なステップです。以下に詳述するアプローチは、皮膚やマラリア原虫にDLN炎症反応を評価するための信頼性の高いアッセイを提供し、様々な実験系に拡張することができます。

<table>
	<tbody>
		<tr>
			<td>Ketamine: Imalgene&#174; 1000</td>
			<td>Merial</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Xylazine: Rompun&#174; 2%</td>
			<td>Bayer</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoFil syringe + 35 gauge needle</td>
			<td>World Precision Instruments&#160;</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Omnican&#174; 50 Insulin syringe 0,5 ml/50 I.U.</td>
			<td>B. Braun Medical&#160;</td>
			<td>9151125</td>
			<td>-</td>
		</tr>
		<tr>
			<td>MultiwellTM 6 well tissue culture plate &#8211; Flat Bottom</td>
			<td>BD Falcon&#160;</td>
			<td>353046</td>
			<td>-</td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer&#160;</td>
			<td>BD Falcon&#160;</td>
			<td>352350</td>
			<td>-</td>
		</tr>
		<tr>
			<td>2 ml syringe</td>
			<td>Terumo</td>
			<td>SS-02S</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BLUE MAXTM 15ml Polypropylene conical tube</td>
			<td>BD Falcon&#160;</td>
			<td>352097</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BLUE MAXTM 50ml Polypropylene conical tube</td>
			<td>BD Falcon&#160;</td>
			<td>352098</td>
			<td>-</td>
		</tr>
		<tr>
			<td>5ml Polystyrene Round-Bottom Tube with 35&#181;m Cell-Strainer Cap</td>
			<td>BD Falcon&#160;</td>
			<td>352235</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS 1X Cacl2- and MgCl2-free</td>
			<td>Life Technologies</td>
			<td>14190-094</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DMEM 1X + GlutaMAXTM</td>
			<td>Life Technologies</td>
			<td>31966-021</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium histolyticum, Type IV 0.5-5.0 FALGPA units/mg solid&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>C5138</td>
			<td>400 U/ml&#160;</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas, type IV&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D5025</td>
			<td>50 &#181;g/ml</td>
		</tr>
		<tr>
			<td>EDTA disodium salt</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>E-5134</td>
			<td>10mM or 2.5 mM</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Biowest</td>
			<td>S1810-500</td>
			<td>-</td>
		</tr>
		<tr>
			<td>HEPES buffer solution (1M)</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td>25 mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue Stain (0,4%)</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td>Dilution 1:10&#160;</td>
		</tr>
		<tr>
			<td>Anti-mouse CD16/CD32 (2.4G2 clone)</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td>10&#181;g/ml final (1:50)</td>
		</tr>
		<tr>
			<td>DAPI FluoroPureTM grade</td>
			<td>Life Technologies</td>
			<td>D21490</td>
			<td>1&#181;g/ml final</td>
		</tr>
		<tr>
			<td>Anti-mouse CD45 (30-F11 clone)</td>
			<td>BD Biosciences</td>
			<td>559864</td>
			<td>&#160;Dilution 1:200</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b (M1/70 clone)</td>
			<td>BD Biosciences</td>
			<td>557657</td>
			<td>&#160;Dilution 1:400</td>
		</tr>
		<tr>
			<td>Anti-mouse CD8&#945; (5H10 clone)</td>
			<td>Life Technologies</td>
			<td>MCD0830</td>
			<td>&#160;Dilution 1:100</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Female C57BL/6JRj mice (7-week-old)&#160;</td>
			<td>Janvier Laboratories</td>
			<td>-</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

myeloid cell isolation from mouse skin and draining lymph node following intradermal immunization with live attenuated plasmodium sporozoites

マラリア原虫のスポロゾイト段階は、蚊に刺さを介して哺乳動物宿主の皮膚に接種されたときにマラリア感染が開始されます。非常に運動性の寄生虫は、肝細胞に侵入し、赤血球感染形式に変換するための肝臓に到達していないだけ。それはまた、皮膚に、それが認識され、居住者及び/又は動員骨髄系細胞によって分解することができる注射部位を、排出近位リンパ節に移動します。生体内イメージングは、明るい蛍光のLys-GFP陽性の皮膚中の白血球および排出リンパ節におけるスポロゾイトおよびCD11c +細胞間の相互作用の早期動員を報告しました。ここでは、回復特定し、マウスの皮膚に動員し、マウスモデルにおいてスポロゾイトの投与量を免疫の皮内注射後のリンパ節を排出している骨髄細胞サブセットを列挙するための効率的な手順を提示します。表現型の特徴用いたマルチパラメトリックフローサイトメトリー提供しますマラリア原虫感染に対する炎症反応の初期にダイナミックな細胞変化を評価するための信頼性の高いアッセイ。

我々は最近、Pの免疫化用量の針注射器注入を実証しましたマウス皮膚におけるベルゲイのスポロゾイトは、皮膚やDLN 19における炎症性単球が続く多形核好中球の連続的な動員を誘導します。プロトコルセクションは、耳の真皮（図1及び2）でスポロゾイトの多数の複数回の注射以下の両方の組織からのライブ骨髄細胞を?...

Watch this Scientific Journal Video about Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites at JoVE.com

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

弱毒で皮内免疫後のマウスの皮膚及び排出リンパノードから骨髄性細胞の単離<em&gt;マラリア原虫</em&gt;スポロゾイト

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

myeloid-cell-isolation-from-mouse-skin-draining-lymph-node-following

Universidad de Cartagena UDEC

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JoVE Journal

Immunology and Infection

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

17.1K ビュー.  Institut Pasteur. We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

ビデオ: Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

鼠径リンパ節の生体顕微鏡

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

免疫・感染学

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

フローサイトメトリーによる肺および肺リンパ節の分析とマウスの非外科的気管内注入

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

広視野と共焦点顕微鏡によるマウスリンパ節スライスにおけるT細胞のex vivoでのイメージング

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Murine Superficial Lymph Node Surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

マウスの表在性リンパ節手術

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

マウス膝窩リンパ節の生体内イメージング

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

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 ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Isolation of Murine Lymph Node Stromal Cells

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

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

ネズミリンパ節間質細胞の単離

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

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

表現型分析のためのヒト皮膚からリンパ球単離と<em&gt; ex vivoで</em&gt;細胞培養

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

酵素消化および勾配分離を使用したマウス皮膚から浸潤白血球の単離

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

弱毒で皮内免疫後のマウスの皮膚及び排出リンパノードから骨髄性細胞の単離

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