この作品は、NIHの助成金P01 AI 068730（SNC、NS）、NIHの助成金CA016520 / TAPITMAT（NS）、Claneil財団（NS）、および卵巣SPORE助成金からFCCCへの民間資金とペンシルベニア大学（によってサポートされていましたP50 CA83638）とフォックスチェイスがんセンターコアグラント（P30 CA06927）（DCC）。著者らは、ペンシルベニア癌の大学で博士G. Danet -デスノイヤーズ監督幹細胞および異種移植片のコアからペンシルベニア州、アンソニーSecretoの大学で博士EJ Delikatny監督光/生物発光の中核施設の優秀な技術支援に感謝手術を支援するためのペンシルベニア州/ OCRC大学の同所性の注入法とDenada DangajへのトレーニングSNCのためのセンター。

1。細胞の文化<ol><li>同所性注入の前に、T175フラスコにDR26腫瘍由来の培養MOV1 キャット細胞 、、彼らは90％コンフルエントになるまで。 1または2 T175フラスコを必要とする注射1〜5万セルを、使用する予定。 </li><li>注射の日に、細胞を収穫し、血球計算盤を用いて細胞数を決定します。 </li><li>一度細胞濃度は、室温で300 × gで5分間、遠心分離によって細胞がペレット、決定されている。 </li><li>スピンに続いて、0.002 M EDTAを滅菌PBSの10マイクロリットルで100万人を持っている細胞を再懸濁</li></ol> 2。手術前<ol><li>手術前に、PBS EDTAの10マイクロリットルで100万MOV1 - キャット細胞と3/10ccインスリン注射器をいっぱいに。 </li><li>加熱パッドにイソフルラン麻酔マウスを転送します。目の脱水を防ぐために、眼軟膏を追加。その後、すぐに手術中麻酔を提供するためにイソフルラン気化器に接続されたノーズコーンのシステムに動物の頭を挿入する。 </li><li>アルコール綿棒で注射部位の消毒した後、皮下3/10ccインスリン注射器で、ケトプロフェン、術前鎮痛剤の5 mg / kgを注入する。 </li><li>バリカンを使用して、動物の尾の基部に胸腰椎接合部から背側の左尾部を削る。完全に毛を除去する除毛クリームを適用します。その後、ぬれたペーパータオルで余分を削除してください。 </li><li>髪が削除されると、ポビドンヨードおよびアルコール綿棒で剃毛面積を滅菌する。その後、切開の周辺手術ドレープを置きます。 </li></ol> 3。手術<ol><li>ただ手術前に、1.5％イソフルレン気化器のレベルを調整します。 </li><li>動物の足のパッドをつまんで完全に麻酔していることを確認します。 </li><li>次に、皮膚の下に脾臓を見つけます。その後、外科はさみを使用すると、脾臓の右上に1〜2センチの背側切開を行います。 </li><li>後腹膜の解剖。マウスの卵巣を取り囲むパッドが観察されます。マウスの卵巣を取り囲む脂肪パッドを把握し、公開する湾曲鉗子を使用してください。 </li><li>滅菌PBSを数滴と水和物器官。 </li><li>位置...撤回、把握することが曲がったピンセットを使用して、...して、注射のため卵巣を確保する</li><li>しっかりと鉗子で卵巣をつかんで、卵巣にMOV1 キャット腫瘍細胞の10マイクロリットルを注入。しっかり理解し、流体逆流や漏れを防止します。 </li><li>すぐに注射後、鉗子によって発揮される張力を解放する。卵巣の穿孔は、自発的に撤回し、閉じる必要があります。 </li><li>針に接続されている吸収性ポリグリコール酸縫合糸を使用して、レトロ腹膜傷を閉じます。 </li><li>ノーズコーンから動物を離します。 </li><li>皮膚をストレッチし、組織接着剤を数滴で背傷のエッジを封印。 </li><li>最後に、経口で動物に抗生物質を100マイクロリットルを管理する。その後回復のためのケージやモニターでそれを取り付けなおします。一週間のために飲料水における抗生物質の動物を保管してください。 </li></ol> （4）in vivoイメージング <ol><li> MOV1 キャット腫瘍細胞の同所性注入後一週間は、in vivoイメージングで行います。イメージング室にイソフルラン麻酔マウスを転送することから始めます。 </li><li> 2％イソフルレン気化器のレベルを下げてください。 </li><li>イメージングシステムの製造元の指示に従ってin vivoイメージングで実行します。このビデオでは、ルミナのシステムが使用されます。 </li><li>画像に、リビングの画像ソフトウェアのデスクトップのアイコンをクリックします。その後、IVISの取得コントロールパネルで、&quot;初期化&quot;を選択します。本器の設定は、カメラの設定に類似しています</li><li>買収のコントロールパネルに、測定器の測定パラメータを設定します。蛍光については、&quot;蛍光灯&quot;をオンにします。各画像と写真を取得するために写真のボックスをクリックします。 </li><li>次に、Autoとして露光時間を設定します。 &quot;ピクセルビニングまたはCCDの解像度&quot;の下に、&quot;ミディアム&quot;をチェック。その後、F /停止や開口の下で、2の値を確認してください。次に、535励起フィルターおよびDsRed発光フィルターを選択します。 </li><li>視野の下に、画像の単一のマウスのためにビューBをクリックしてください。 </li><li>その後、画像取得を開始するために取得するをクリックしてください。 </li><li>画像の取り込みが完了すると、関心領域、またはROI、信号の測定するツールを使用してください。信号の値と領域を解放するために、測定のアイコンをクリックしてください。 </li><li>最後に、ユーザーのフォルダに画像を保存する&quot;保存&quot;をクリックしてください</li><li>画像が保存された後、イソフルランの投与を中止し、そのケージにマウスを返します。マウスはすぐに目を覚ます必要があります。 </li></ol> 5。代表的な結果 - 使用してこのプロトコルを、同所性卵巣癌のin vivo増殖非侵襲的な手順を使用して、少なくとも3週間は監視することができます。 <img src="/files/ftp_upload/2146/2146fig1.jpg" alt="図1" /> 図1。ネガティブコントロールとしてMOV1 キャット細胞 、またはPBSは、同所。非腫瘍傾向のあるマウス（それぞれ右動物と左動物と）の卵巣に注入し、in vivoイメージングでは 2週間後に行われた。卵巣の生着MOV1 キャット細胞によって生成された蛍光発光を測定し、陰性対照マウスからのそれと比較した。 <img src="/files/ftp_upload/2146/2146fig2.jpg" alt="図2" /> 図2。MOV1 キャットの卵巣腫瘍の凍結切片をモノクローナル抗体は、腫瘍浸潤リンパ球（黒い矢印）を検出するためのDAB基質（ダークブラウン）に続いてビオチン化抗- CD4で染色した。細胞は、細胞核（青）を可視化するためにメチルグリーンで対比染色した。腫瘍細胞は、（赤矢印）、T細胞からの形態学的に異なるいた。スライドは、40倍拡大表示されます。

<ol>
	<li>Etzioni, 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=The+case+for+early+detection.">The case for early detection.</a> Nat Rev Cancer. 3, 243-252 (2003).</li><li>Sasaroli, D., Coukos, G., Scholler, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+CA125:+the+coming+of+age+of+ovarian+cancer+biomarkers.+Are+we+there+yet.">Beyond CA125: the coming of age of ovarian cancer biomarkers. Are we there yet.</a> Future Medicine. 3, 275-288 (2009).</li><li>Anderson, G. 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=Assessing+Lead+Time+of+Selected+Ovarian+Cancer+Biomarkers:+A+Nested+Case+Control+Study.">Assessing Lead Time of Selected Ovarian Cancer Biomarkers: A Nested Case Control Study.</a> J Natl Cancer Inst. 102, 26-38 (2010).</li><li>Auersperg, N., Wong, A. S., Choi, K. C., Kang, S. K., Leung, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+surface+epithelium:+biology,+endocrinology,+and+pathology.">Ovarian surface epithelium: biology, endocrinology, and pathology.</a> Endocr Rev 22. , 255-288 (2001).</li><li>Dubeau, 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+cell+of+origin+of+ovarian+epithelial+tumours.">The cell of origin of ovarian epithelial tumours.</a> Lancet Oncol. 9, 1191-1197 (2008).</li><li>Connolly, D. 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=Female+mice+chimeric+for+expression+of+the+simian+virus+40+TAg+under+control+of+the+MISIIR+promoter+develop+epithelial+ovarian+cancer.">Female mice chimeric for expression of the simian virus 40 TAg under control of the MISIIR promoter develop epithelial ovarian cancer.</a> Cancer Res. 63, 1389-1397 (2003).</li><li>Hensley, 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=Magnetic+resonance+imaging+for+detection+and+determination+of+tumor+volume+in+a+genetically+engineered+mouse+model+of+ovarian+cancer.">Magnetic resonance imaging for detection and determination of tumor volume in a genetically engineered mouse model of ovarian cancer.</a> Cancer Biol Ther. 6, (2007).</li><li>Deliolanis, N. 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=Performance+of+the+red-shifted+fluorescent+proteins+in+deep-tissue+molecular+imaging+applications.">Performance of the red-shifted fluorescent proteins in deep-tissue molecular imaging applications.</a> J Biomed Opt. 13, (2008).</li><li>Hoffman, R. 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+better+fluorescent+protein+for+whole-body+imaging.">A better fluorescent protein for whole-body imaging.</a> Trends Biotechnol. 26, (2008).</li><li>Shcherbo, D., 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=far-red+fluorescent+protein+for+whole-body+imaging.">far-red fluorescent protein for whole-body imaging.</a> Nat Methods. 4, 741-746 (2007).</li><li>Fu, X., Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+ovarian+carcinoma+metastatic+models+constructed+in+nude+mice+by+orthotopic+transplantation+of+histologically-intact+patient+specimens.">Human ovarian carcinoma metastatic models constructed in nude mice by orthotopic transplantation of histologically-intact patient specimens.</a> Anticancer Res. 13, 283-286 (1993).</li><li>Kiguchi, 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=A+patient-like+orthotopic+implantation+nude+mouse+model+of+highly+metastatic+human+ovarian+cancer.">A patient-like orthotopic implantation nude mouse model of highly metastatic human ovarian cancer.</a> Clinical &amp; experimental metastasis 16. , 751-756 (1998).</li><li>Bao, 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=Activation+of+cancer-specific+gene+expression+by+the+survivin+promoter.">Activation of cancer-specific gene expression by the survivin promoter.</a> J Natl Cancer Inst. 94, 522-528 (2002).</li><li>Connolly, D. C., Hensley, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenograft+and+Transgenic+Mouse+Models+of+Epithelial+Ovarian+Cancer+and+Non+Invasive+Imaging+Modalities+to+Monitor+Ovarian+Tumor+Growth+In+situ+-Applications+in+Evaluating+Novel+Therapeutic+Agents.+Current+Protocols+in+Pharmacology+45.">Xenograft and Transgenic Mouse Models of Epithelial Ovarian Cancer and Non Invasive Imaging Modalities to Monitor Ovarian Tumor Growth In situ -Applications in Evaluating Novel Therapeutic Agents. Current Protocols in Pharmacology 45.</a> , 1-36 .</li><li>Milne, 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=Systematic+analysis+of+immune+infiltrates+in+high-grade+serous+ovarian+cancer+reveals+CD20,+FoxP3+and+TIA-1+as+positive+prognostic+factors.">Systematic analysis of immune infiltrates in high-grade serous ovarian cancer reveals CD20, FoxP3 and TIA-1 as positive prognostic factors.</a> , (2009).</li></ol>

利害の衝突は宣言されません。

手術と同所注射卵巣嚢における同所性注入は、トレーニングと精度が要求されます。このように<ol><li>貧しい手術経験の場合、最初の死体との練習。 </li><li>彼らは未経産の雌と比較して注入し、増加の生存を容易に時間をかけて大きな卵巣を開発するように優先的に初産の女性を（1つまたは2つのリットル）を使用してください。 </li><li>マウス卵巣嚢の小さなサイズの?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM-GLUTAMAX</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10564-011</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14040</td>
<td>&nbsp;</td>
<tr>
<td>Versene</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>17-711E</td>
<td>&nbsp;</td>
<tr>
<td>Heating pad</td>
<td>&nbsp;</td>
<td>Deltaphase</td>
<td>39 DP</td>
<td>&nbsp;</td>
<tr>
<td>Povidone pads</td>
<td>&nbsp;</td>
<td>Dynarex</td>
<td>1108</td>
<td>&nbsp;</td>
<tr>
<td>Alcohol pads</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>06-669-62</td>
<td>&nbsp;</td>
<tr>
<td>Artificial tears ointment </td>
<td>&nbsp;</td>
<td>Phoenix Pharma., Inc.</td>
<td>17845-153</td>
<td>&nbsp;</td>
<tr>
<td>Ketoprofen</td>
<td>&nbsp;</td>
<td>Fort Dodge laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>3cc/insulin syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309301</td>
<td>&nbsp;</td>
<tr>
<td>Polyg Polyglycolic Acid suture/needle (3/8 19mm)</td>
<td>&nbsp;</td>
<td>Syneture</td>
<td>9612-31</td>
<td>&nbsp;</td>
<tr>
<td>Tissue adhesive</td>
<td>&nbsp;</td>
<td>Vetbond</td>
<td>3M</td>
<td>&nbsp;</td>
<tr>
<td>Vet Bactrim/ oral suspension</td>
<td>&nbsp;</td>
<td>Hi-tech Pharmacal</td>
<td>840823</td>
<td>&nbsp;</td>
<tr>
<td>IVIS-Lumina</td>
<td>&nbsp;</td>
<td>Caliper lifesciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isofluorane</td>
<td>&nbsp;</td>
<td>Phoenix Pharma., Inc.</td>
<td>J108013</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum, Qualified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10437036</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>TurboFP635 mammalian vector</td>
<td>&nbsp;</td>
<td>Evrogen</td>
<td>FP721</td>
<td>&nbsp;</td>
<tr>
<td>T175 flasks</td>
<td>&nbsp;</td>
<td>cellstar</td>
<td>660-190</td>
<td>&nbsp;</td>		</tbody>
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an orthotopic model of serous ovarian cancer in immunocompetent mice for in vivo tumor imaging and monitoring of tumor immune responses

背景：卵巣がんは一般的にケース/致死率が高いため、米国の女性は1,2,3の間で、すべての婦人科悪性腫瘍の最も致命的なまま進行した段階で診断されます。漿液性腫瘍は卵巣癌の最も広く普及している形ですと4,5 TG - MISIIR - TAGトランスジェニックは、自発的に腫瘍のこのタイプを開発する唯一のマウスモデルを表しています。ミュラー阻害物質II型受容体（MISIIR）遺伝子のプロモーター6の制御下TG - MISIIR - TAGマウス急行SV40形質転換する地域。追加のトランスジェニック系統は、発現するSV40タグの導入遺伝子を同定されているが、卵巣腫瘍を発症しない。非腫瘍傾向のあるマウスは、C57BL / 6マウスの一般的な寿命を示し、肥沃です。これらのマウスは、TG - MISIIR - TAG - DR26マウスから単離した腫瘍細胞に対する同系移植のレシピエントとして使用することができます。 目的：腫瘍イメージングは7可能ですが、深い腫瘍の早期発見が小さい生きている動物に挑戦する。漿液性卵巣癌の免疫学的に無傷の動物モデルにおける前臨床試験を有効にするには、我々はin vivoイメージング、腫瘍微小環境と腫瘍免疫応答の研究で可能に卵巣癌のこのタイプの同系マウスモデルを説明します。 方法：我々は最初の26週齢のDR26 TG - MISIIR - TAGの女性で収穫された自発的な卵巣腫瘍からTAG +マウスの癌細胞株（MOV1を）派生。その後、我々は安定Katushka、635分の588 nmの8,9,10での励起/発光極大を持つイソギンチャクEntacmaea quadricolorから赤色蛍光タンパク質の遠赤変異体をコードTurboFP635レンチウイルス哺乳類ベクターとMOV1細胞を形質導入。我々は、同所の非腫瘍が発生しやすいTG - MISIIR - TAG雌マウスの卵巣11,12,13,14でMOV1 キャットを注入。腫瘍の進行は 、in vivo光イメージングおよび腫瘍微小環境で続いていた免疫組織化学により解析した。 結果：同所移植MOV1 キャット細胞は漿液性卵巣腫瘍を開発した。 MOV1 キャットの腫瘍は最大3週間注入後in vivoイメージングで可視化することができる（図1）とのようなヒト卵巣癌15（図2）で観察された白血球、で浸潤した。 結論：我々は、深部組織のKatushkaの高いpH安定性と光安定性のために早期の腫瘍のin vivoイメージングに適した卵巣癌の同所性モデルを記述する。我々は、腫瘍免疫応答と免疫療法のin vivoイメージングの研究とモニタリングのための漿液性卵巣癌のこの小説同系モデルの使用を提案する。

Watch this Scientific Journal Video about An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses at JoVE.com

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

勉強する<em&gt; in vivoで</em&gt;腫瘍の増殖と腫瘍微小環境、我々は、免疫動物の卵巣癌の同系と同所性マウスモデルを使用。我々はKatushka蛍光タンパク質（MOV1でマウスの腫瘍細胞株（MOV1）を形質導入<sup&gt; KAT</sup&gt;）と、ここでは、卵巣での同所移植を示し、<em&gt; in vivoで</em&gt;イメージング。

のための免疫担当マウスにおける漿液性卵巣癌の同所性モデル in vivoで</em腫瘍免疫応答の>腫瘍イメージングとモニタリング

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

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Research

JoVE Journal

Immunology and Infection

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

18.1K ビュー.  University of Pennsylvania-School of Medicine. 勉強する in vivoで腫瘍の増殖と腫瘍微小環境、我々は、免疫動物の卵巣癌の同系と同所性マウスモデルを使用。我々はKatushka蛍光タンパク質（MOV1でマウスの腫瘍細胞株（MOV1）を形質導入 KAT）と、ここでは、卵巣での同所移植を示し、 in vivoでイメージング。

ビデオ: An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

B細胞のCD40 -活性化のモデルマウス

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

免疫・感染学

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

アン<em&gt;インビトロ</em&gt; HIV重感染のコンテキストでのマラリアに対する免疫応答を測定するためのモデル

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

IL-8トランジェントトランスジェニックマウスモデル<em&gt;インビボ</em&gt;炎症反応の長期モニタリング

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

サルモネラの分離-大食細胞のファゴゾームを含む

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

蛍光寿命イメージングによる細胞内のスネアを可視化

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

腫瘍特異的 CD8+ T 細胞応答における Immunodominance を研究するためのインビボ細胞毒性アッセイ

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

アチミックマウスにおけるヒト胸膜中皮腫の正形細胞モデルのPET/CTによる移植とモニタリング

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

腫瘍微小環境における免疫細胞集団の可視化、定量化、およびマッピング

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

マウスにおける神経免疫ダイナミクスの反復インビボイメージングを行う正確な脳マッピング

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

in vitro腫瘍微小環境における免疫応答を解剖するためのマイクロ流体共培養モデル

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

のための免疫担当マウスにおける漿液性卵巣癌の同所性モデル in vivoで腫瘍イメージングとモニタリング

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