この作品は、博士ジム胡にCIHRとCFカナダの補助金によっておよびCFカナダラーフルKushwahに授与博士課程の学生の身分によって資金を供給された。

1。オボアルブミンとマウスの作<ol><li> OVA（グレードV;シグマ、MO）の溶液を調製し、1 mg / mlの濃度で滅菌したPBS（溶液は-80℃で保存することができます）。 </li><li> OVA-ミョウバン混合物を調製するためには、チューブにミョウバンを取ると1:1の割合でチューブをボルテックスしながら滴下する方法でOVAソリューションを追加します。 30分間混合物を攪拌して混合した後すぐに使用します。 </li><li> 1 mlのシリンジを使用して、マウス腹腔内に混合物を0.2ミリリットルを注入し、2週間後に再度注入を繰り返します。 </li></ol> 2。 FITC標識アルブミンを持つマウスの鼻腔内チャレンジ<ol><li> 7日OVA-ミョウバンの第二の注入後、マウスはOVA-FITCを鼻腔内に挑戦する準備ができました。 </li><li> -80℃でアリコートでは1 mg / mlと店の濃度で滅菌したPBSを使用して、OVA-FITC溶液（シグマ）℃を準備します。 </li><li> 50mlファルコンチューブの底に、化学hの滅菌ガーゼを置くOODは、ガーゼの上にAerraneの5ミリリットルを追加します。マウスを麻酔するためには、約5〜10秒間チューブに動物を指示します。 </li><li>直立した状態で麻酔した動物を保持し、滅菌したピペットチップを使用して、ドロップダウン賢明な方法でマウスの鼻孔の上にOVA-FITC溶液を100μL添加します。 </li><li>次の二日間OVA-FITCと鼻の挑戦を繰り返します。 </li></ol> 3。肺と所属リンパ節からの単細胞懸濁液の調製<ol><li> Euthanylの腹腔内注射を使用してマウスを生け贄に捧げる。 </li><li> DC解析を複雑にする可能性がマクロファージの汚染を減らすために、肺洗浄​​が行われます。肺を洗浄するには、マウス気管を簡単にカテーテルと注射器を露出し、カニューレを挿入されています。5mMのEDTAを含む氷冷PBSを使用して、下気道が肺胞腔からセルを削除するには、3回洗浄されています。 </li><li> 10 U / mlの肝臓を含むPBS 10mlで肺を灌流心臓の右心室経由で、肺の血管から血液細胞を除去します。肺は血液細胞の大部分の除去を示す、白くなるまでperfusionsを実行します。これは、末梢血からの汚染細胞を除去することが特に重要です。 </li><li>肺を解剖し、縦隔リンパ節（MLN）を取り外し、別の皿のリンパ節を配置します。 （FITC蛍光の消光を防ぐために点灯する組織の露出を最小限に抑えるようにしてください。） </li><li>ペトリ皿に肺を置き、はさみを使用してミンチ。次の37℃で25分間、肺、消化°C、250 U / mlのコラゲナーゼD（Roche）を使用して、コラゲナーゼ活性を停止するには、次の5分間のEDTAを（10mMの最終濃度）を追加します。 </li><li> 100μmのセルストレーナーを通して消化され、肺の断片を渡すと、赤血球を除去するために低張性溶解を実行します。単一の細胞懸濁液は、DCのさらなる分析のために準備ができています。 </li><li> MLNから単一細胞懸濁液を調製するためには、t微細な針を使用してMLNを容易にし、セルストレーナーを通して緊張が続く上記のようにコラゲナーゼで消化する。 </li></ol> 4。成熟/移行を評価するためのDCマーカーの染色<ol><li>肺の樹状細胞を識別するために、染色のCD11cおよびCD11bのために実行されます。さらに、成熟のために評価するために、染色はDCが成熟するようにアップレギュレートされるCD86とCD80に対して実行されています。のCD11c + CD11bを+ OVA-FITC + MLN中の細胞をOVA気道チャレンジに応答して、肺からMLNへの移行肺樹状細胞として識別されます。 </li><li> FACS緩衝液（1％FBSおよび1mMのEDTAを含むPBS）、アリコートFACS染色のための細胞懸濁液/チューブ150μlの10×10 6細胞/ mlの濃度で細胞をサスペンドします。肺樹状細胞の同定については、次の汚れは、必要とされる。染色コントロール（OVA-FITCにさらされていないマウスから肺細胞）、OVA-FITC制御、CD11cは単一の制御、CD11bを単一の制御を、CD11bを+のCD11c +ダブル制御、CD11bを+ OVA-FITC +二重制御は、CD11c + OVA-FITC +ダブル制御、MHC II OVA-FITC +のCD11c + CD11bを+ MHCIIを染色し、単一の制御、CD86、単一の制御、CD80単一のコントロールとサンプル+ 、OVA-FITC +のCD11c + CD11bを+ CD86 +とOVA-FITC +のCD11c + CD11bを+ CD80 +。 </li><li> 、DCの移行を評価するMLNからの単一細胞懸濁液の絶対細胞数を実行し、その後、以下のチューブを準備するには、次のCD11c染色コントロール（OVA-FITCにさらされていないマウスからのMLN細胞）、OVA-FITC制御、+単一のコントロール、CD11bを+二重制御、CD11bを+ OVA-FITC +二重制御は、CD11c + OVA-FITC +ダブルコントロールおよびCD11c + CD11bを+ OVA-FITC +のサンプル。 </li></ol> 5。代表的な結果気道アレルギー性炎症を誘導するために腹腔内感作に必要な時間はポイントが重要であり、 図1に示すように実施すべきである。気道アレルギー性炎症の誘導を確認するために、腹腔内感作と気道のOVAチャレンジ後、いくつかのマウスを屠殺することができ、 図2に示すように、組織学的分析は、肺切片上で行うことができます。炎症性細胞の存在は、ヘマトキシリン·エオシン染色により確認することができる（ 図2A）と粘液産生の存在は、定期的な酸シッフ染色（ 図2B）によって評価することができる。完全にこれは、OVA感作マウス8のOVAチャレンジ後の気道アレルギー性炎症の誘導を確認します。対照的に、生理食塩水処置したマウスからの肺のセクションは任意の粘液産生の有無とともに、任意の炎症の自由になることが期待されている。さらに、以下のOVAチャレンジ、肺樹状細胞unde<html> rgo成熟および所属リンパ節への後続の移行。感作とOVAでチャレンジしたマウスからの縦隔リンパ節（MLN）におけるCD11c +細胞の解析は、生理食塩水で処置したマウス（ 図3A）と比較して、CD11c +細胞の割合が高いを示すことが期待されています。また、OVA感作とチャレンジしたマウスのMLNにおけるのCD11c + CD11bを+ OVA-FITC +細胞の絶対細胞数の解析は、生理食塩水処置したマウスからの対応という有意に高い（数倍高い、すなわち）のカウントを示すことが期待される（ 図3B）。 <img alt="図1" src="/files/ftp_upload/4014/4014fig1.jpg" /> 図1。FITC標識した卵白アルブミン（OVA-FITC）と気道のチャレンジと一緒にマウスにオボアルブミン（OVA）誘発されるアレルギー性気道炎症の誘導のための実験プロトコル。 fig2.jpg &quot;/&gt; OVA気道チャレンジに続いて図2 OVA感作は、（A）に示すように、肺切片のヘマトキシリン·エオシン染色により同定などの気道アレルギー性炎症の誘導につながり、また、肺の過ヨウ素酸シッフ染色によって識別される気道の粘液産生につながる（B）に示すようにセクションを参照してください。 <img alt="図3" src="/files/ftp_upload/4014/4014fig3.jpg" /> 図3。OVA-FITCの課題は、肺からの縦隔リンパ節（MLN）にDCへの移行を誘導する。 （A）フローサイトメトリープロットはコントロールまたはOVA感作マウスのMLNにCD11c +細胞の割合を描いた。 （B）生理食塩水またはOVA感作マウスのMLNでのCD11c + CD11bを+ OVA-FITC +細胞の絶対数。 * P &lt;0.05。 <a href="http://www.jove.com/files/ftp_upload/4014/4014fig3large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。

<ol>
	<li>Kushwah, R., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+dendritic+cells+in+the+induction+of+regulatory+T+cells.">Role of dendritic cells in the induction of regulatory T cells.</a> Cell Biosci. 20, (2011).</li><li>Kushwah, R., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexity+of+dendritic+cell+subsets+and+their+function+in+the+host+immune+system.">Complexity of dendritic cell subsets and their function in the host immune system.</a> Immunology. 133, 409-419 (2011).</li><li>Lambrecht, B. N., Hammad, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+our+breath+away:+dendritic+cells+in+the+pathogenesis+of+asthma.">Taking our breath away: dendritic cells in the pathogenesis of asthma.</a> Nat Rev Immunol. 3, 994-1003 (2003).</li><li>Hammad, H., Lambrecht, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+epithelial+cells:+linking+innate+and+adaptive+immunity+in+asthma.">Dendritic cells and epithelial cells: linking innate and adaptive immunity in asthma.</a> Nat. Rev. Immunol. 8, 193-204 (2008).</li><li>Lloyd, C. M., Murdoch, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerizing+allergic+responses+in+the+lung.">Tolerizing allergic responses in the lung.</a> Mucosal. Immunol. 3, 334-344 (2010).</li><li>Herz, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+of+murine+animal+models+to+study+the+development+of+allergic+bronchial+asthma.">The relevance of murine animal models to study the development of allergic bronchial asthma.</a> Immunol. Cell Biol. 74, 209-217 (1038).</li><li>Herz, U., Lumpp, U., Daser, A., Gelfand, E. W., Renz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+animal+models+to+study+the+central+role+of+T+cells+in+immediate-type+hypersensitivity+responses.">Murine animal models to study the central role of T cells in immediate-type hypersensitivity responses.</a> Adv. Exp. Med. Biol. 409, 25-32 (1996).</li><li>Nakae, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF+can+contribute+to+multiple+features+of+ovalbumin-induced+allergic+inflammation+of+the+airways+in+mice.">TNF can contribute to multiple features of ovalbumin-induced allergic inflammation of the airways in mice.</a> J. Allergy Clin. Immunol. 119, 680-686 (2007).</li><li>Guth, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+environment+determines+unique+phenotype+of+alveolar+macrophages.">Lung environment determines unique phenotype of alveolar macrophages.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 296, L936-L946 (2009).</li><li>Kushwah, R., Cao, H., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+pulmonary+T+cell+response+to+helper-dependent+adenoviral+vectors+following+intranasal+delivery.">Characterization of pulmonary T cell response to helper-dependent adenoviral vectors following intranasal delivery.</a> J. Immunol. 180, 4098-4108 (2008).</li><li>Kushwah, R., Oliver, J. R., Zhang, J., Siminovitch, K. A., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+dendritic+cells+induce+tolerance+in+mice+through+suppression+of+dendritic+cell+maturation+and+induction+of+antigen-specific+regulatory+T+cells.">Apoptotic dendritic cells induce tolerance in mice through suppression of dendritic cell maturation and induction of antigen-specific regulatory T cells.</a> J. Immunol. 183, 7104-7118 (2009).</li></ol>

利害の衝突が宣言されません。

ここで紹介する方法は、気道チャレンジOVA-FITCの配信に基づいて、肺樹状細胞を分析するためのフローサイトメトリーベースのアプローチを提供しています。これは、OVA-FITCを取ると、監視されているので、DC集団は、実質的にOVA誘発性アレルギー性​​気道炎症の過程で気道免疫応答に参加しているものである肺樹状細胞の選択的な監視が可能になります。コントロールマウスでは、アレル?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>オボアルブミン</td><td> Sigma-Aldrich社</td><td> A5503 </td></tr><tr><td>ミョウバン</td><td>サーモフィッシャーサイエンティフィック</td><td> 77161 </td></tr><tr><td> OVA-FITC </td><td> Sigma-Aldrich社</td><td> A9771 </td></tr><tr><td>コラゲナーゼD </td><td>ロシュ社</td><td> 11088858001 </td></tr><tr><td>抗体</td><td>電子バイオサイエンス</td><td></td></tr></tbody></table>

analysis of pulmonary dendritic cell maturation and migration during allergic airway inflammation

樹状細胞（DC）が抗原特異的T細胞を活性化することにより適応免疫応答の開始に関与する重要なプレーヤーである。 DCは、定常状態での末梢組織に存在していますが、抗原刺激に応答して、樹状細胞は、抗原を取り上げ、急速にそれらが抗原1,2に対するT細胞応答を開始する所属リンパ節に移行します。さらに、樹状細胞はまた、自己免疫などアレルギー性免疫応答3を開始するのに重要な役割を果たしている。 DCは、肺の環境4の設定に公差の免疫応答と誘導の両方の開始に重要な役割を果たしています。肺の環境では、環境抗原5の広大な配列への暴露により主に寛容です。ただし、一部の個体でアレルギーや喘息の誘発につながる耐性の休憩がある。本研究では、気道DCの成熟とMIGを監視するために使用することができます戦略を記述感作に用いた抗原に応答して配給。気道DCの成熟と移行の測定は、気道アレルギー性​​炎症時に免疫応答の動態の評価を可能にし、また根本的なメカニズムとともに、その後の免疫応答の大きさを理解する上で役立ちます。 我々の戦略は、増感剤としてオボアルブミンの使用に基づいています。オボアルブミン誘発性アレルギー性喘息は、気道好酸球増加、肺の炎症、喘息6,7中に見つかった上昇IgE値を再現するために広く使われているモデルです。感作後、マウスを取り込みオボアルブミン気道樹状細胞の特異的な標識を可能にFITC標識アルブミンの鼻腔内送達によって、求められています。次に、いくつかのDC特異的なマーカーを使用して、我々はこれらの樹状細胞の成熟を評価することができ、また、フローサイトメトリーを用いることにより、所属リンパ節への移行を評価することができます。

Watch this Scientific Journal Video about Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation at JoVE.com

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

我々は、オボアルブミン誘発アレルギー性​​気道炎症の設定でオボアルブミンに反応して肺樹​​状細胞の成熟と移行を監視するための戦略について説明します。この戦略は、感染の設定で肺樹状細胞の遊走を評価するために変更することができます。

アレルギー性​​気道炎症時の肺樹状細胞の成熟および移行の解析

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

analysis-pulmonary-dendritic-cell-maturation-migration-during

Research

JoVE Journal

Immunology and Infection

14.9K ビュー.  McMaster University, Hamilton. 我々は、オボアルブミン誘発アレルギー性​​気道炎症の設定でオボアルブミンに反応して肺樹​​状細胞の成熟と移行を監視するための戦略について説明します。この戦略は、感染の設定で肺樹状細胞の遊走を評価するために変更することができます。

ビデオ: Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

移行のヒトTリンパ球の単離、培養および解析インビトロ</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

免疫・感染学

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

細胞の成熟せずに樹状細胞の効率的なトランスフェクションに最適化されたプロトコル

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

マウス大動脈内の免疫細胞のフローサイトメトリー分析

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

使用して樹状細胞の移行の監視<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁気共鳴イメージング

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

急性及び慢性炎症を区別するために生体内イメージング法における

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

マウスの脾臓から不変ナチュラルキラーT細胞を単離および拡張するための最適化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

単離、特徴付けおよび歯肉免疫細胞ネットワークの機能検討

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

の気管内の管理<em&gt;インフルエンザ菌</em&gt;マウスモデルにおいて気道炎症を研究します

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

での感染の間に流入領域リンパ節へのマウスの皮膚樹状細胞の移動を研究するためにCFSEベースアッセイ<em&gt;マイコバクテリウム・ボビス</em&gt;カルメット - ゲラン

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

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

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

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

精密カットライブ肺樹状細胞を可視化するために、マウスの肺スライスを

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

アレルギー性​​気道炎症時の肺樹状細胞の成熟および移行の解析

アレルギー性気道炎症時の肺樹状細胞の成熟および移行の解析