著者は編集の援助のためにアンダロンとピエールルイLoyherに感謝したい、2光子顕微鏡と支援を飼育したマウスのための動物施設「NAC」とカミーユBaudessonの支援についてPlateforme ImageriePitié-Salpêtrière（PICPS）。これらの結果につながる研究グラント契約の下で欧州共同体の第7次フレームワークプログラム（FP7 / 2007から2013）から資金提供を受け、N 304810を°いる - RAIDの、そしてnは241440-Endostem、INSERMから、大学ピエール·マリー·キュリーから &quot;出現を° 「ラから「協会ラルシェルシュシュルルがんを注ぐ」とから「通信社国立·デ·ラ·ルシェルシュ」プログラムの創発2012（ANR-EMMA-050）」から、「リーグコントル·ルがん。 PHはラ &quot;リーグコントル·ル癌」によってサポートされていました。

注：すべての実験プロトコルは、フランスの動物実験や倫理委員会によって承認され、番号A-75から2065に「サービスの保護らサンテAnimales、エンバ」によって検証された。サンプルサイズは、実験の再現性を確実にするために選択され、動物の倫理規定の3Rに応じている。 マウスの作製<ol><li>麻酔<ol><li>イメージングの短時間（1時間未満）のために、ケタミン（100mg / kg）およびキシラジン（10mg / kgの）を含有する生理食塩水200μlを腹腔内注射してマウスを麻酔し。 </li><li>あるいは、（4時間まで）イメージングの長期間適合マスクを介して、O 2 / N 2 Oの70/30混合物で気化イソフルラン2.5％の吸入で、マウスを麻酔し。 </li><li>注：この方法は、より安定した無意識のために提供し、薬のシリアル腹腔内注射を避けることができます。 </lI&gt; <li>マウスを麻酔したら、ニッパーで足パッドの刺激を通じて無意識をチェック。 </li></ol></li><li>血管染色/追加の染色<ol><li>血管の染色：尾静脈に静脈内にローダミン - デキストランを200μl（2 MDA、生理食塩水緩衝液中10mg / ml）を注入する。 </li><li>好中球の染色のために：尾静脈に静脈内に生理食塩水100μl中Ly6G-PE（クローン1A8）2μgのを注入する。 </li></ol></li><li>固定化<ol><li>固定化のために、顕微鏡に及び麻酔ガス吸入器に適合したカスタムメイドの定位ホルダーを使用しています。 注：sterotacticホルダーは、動物の頭を締め、2金属板ブラケット、固定されている1と他のリムーバブルと適応性を持つ金属支持体である。 </li><li>呼吸動きからの分離を確実にするために、保持プレート間のマウスの頭部をインストールします。 SKをストレッチする保持プレートとヘッドと耳を締めインチ安定性と動物の呼吸福祉のために確認してください。 </li></ol></li><li>頭皮を削除<ol><li>慎重に眼との接触を避け、無菌のアプリケーターで頭皮の髪を濡らし、エタノール70％使用しています。 </li><li>完全に前頭骨に頭頂骨の後方から頭皮を削除するには、滅菌ハサミやニッパーで皮膚をカットします。大規模な出血を防ぐために、目と耳から3ミリメートルまで遠ざけること。頭蓋骨（骨膜）をカバー疎性結合組織を削除します。暖かいPBSに浸したペーパータオルで残りの髪を洗ってください。 </li></ol></li><li>液浸システムのセットアップ<ol><li>分布を制御するための小さなゲージの針を用いて頭蓋骨に直接外科的接着剤で直径18mmのゴムリングを貼り付けます。 （30μlのに十分であるべきである）の漏洩を防止するために、ゴムリングの全周を接着。 </li><li> PBSに浸したペーパータオルでリング下に最後にもう一度頭蓋骨をきれいにし、その後の完全な浸漬し、37℃のPBSを追加頭蓋骨。眼の乾燥を避けるために、マウスのそれぞれの眼にNaClの0.9％溶液または特定の眼軟膏のドロップを追加します。目的の下に定位ホルダーをドラッグします。 </li><li>約ダイレクトトランスミッションを介し顕微鏡接眼レンズを使用してフィールドを配置します。 </li></ol></li></ol> 2.二光子イメージングの取得<ol><li>選択的に680から1080ナノメートルまで調節可能で、NIR光の140fsパルスを提供するサファイア結晶レーザ、レーザパワー制御のための音響光学変調器：チタンと結合し、多光子顕微鏡を使用する。 2ダイクロイックミラー（565 nmおよび690 nm）で、610分の565と550分の500バンドパスフィルタの組み合わせ（3蛍光チャネルの同時録画を可能にする）3外部の非デスキャン検出器（NDD）を含む構成を使用して、プランアポクロマート485ショートパスフィルターは、水浸対物レンズ（NA = 1）×20。 </li><li> anaesの良いhomeothermyを可能にするために加熱室を設定thetizedマウス。、 注：温度が、より安定性のためにセッションを画像化する1時間前に設定されることがあります。我々のケースでは、32°Cで麻酔したマウス（36~37℃）の最適な体温を得るために選択され、4時間後まで維持される。この温度は、使用されるシステム（デジタルプローブは、この点を確認するために使用することができる）に適合させることができる。また、使用される加熱チャンバは、黒/暗い、外光の混入を避けるために、システム（目的および電動プレート）の一部を覆う。 </li><li>システムの電源を入れます<ol><li>サファイアレーザー、その後全体の顕微鏡システム：TIの電源をオンにします。取得ソフトウェアを起動します。 870 nmのレーザー波長範囲を設定する。記録のために適切なNDDを選択します。 注：この例では、第二高調波発生（SHG）19と、シアン蛍光タンパク質（ECFP）は485ショートパスフィルタの後NDDによって検出される。 550分の500バンドパスフィルタ及びローダミン - デキストランをtで検出された後ECFPもNDDによって検出される彼NDD 610分の565バンドパスフィルタの後。 </li></ol></li><li>取得の設定を調整<ol><li>ソフトウェアの「ライブ取得」イメージングコマンドを使用し、顕微鏡、方向コントロールを使用して、ECFP及びローダミン信号の領域にフォーカスを調整します。最小限の光損傷を有する最良の信号対雑音比を得るために、最小限にレーザパワーを設定する。 注：使用される電力設定は、関心領域の深さに応じて変化する。典型的には、AOMの設定は、15ミリワットの周りの目的を超えてレーザパワーを表して約20％に設定される。各NDDは、光退色および光損傷を低減することがPMTのゲインに電力を増加させる方がよい。典型的な取得の設定は、単一の1.58マイクロ秒のピクセル滞留時間でスキャンし、1.5倍率512×512ピクセルの解像度である。 </li></ol></li><li>関心領域を選択します<ol><li>記録パラメータが設定されると、再び「ライブ」の取得コマンドを使用リアルタイムで監視すべき所望の（x、y、z）の欄を選択するための組織を調査する。 注：通常、我々は両方の血管および実質領域を含むフィールドを選択しました。 </li><li> 「位置」ソフトウェアコマンドを使用してフィールドを登録します。 注：このコマンドは異なる遠いフィールドのx、y、z座標を記憶する。 </li><li>同じ手順を使用して、（3まで）興味のある追加のフィールドを選択し、登録します。 </li><li>選択した倍率で、必要量に応じて第一記憶した位置にある「zスタック」ソフトウェアコマンドを使用して、3次元zスタックを設定します。最初の深い位置し、次に最も高い位置を記憶します。深さのレーザパワーを正規化する。 注：複数の付随フィールドイメージングのために、我々は12μmの厚さのボリュームを取得するフィールドごとに3μmのZ-段階で区切られた5スライスを獲得する。これらの設定は、研究細胞の種類に適合させることがあります。 </li></ol></li><li>起動買収<ol><li>選択する220と、時系列「ソフトウェアコマンドと時間間隔の持続時間とサイクル数を選択します。 「実験を開始する「ソフトウェアコマンドを選択することで、必要な時間の経過と期間に応じてタイムラプスイメージングを開始します。 注：私たちのケースでは、30分のリアルタイムを表す60サイクルの間に各30秒1のボリュームを取得する。短い時間の経過は、血管内の速い細胞ローリングを追跡するために行うことができる。この場合、薄いzスタックと2以下の異なるフィールドを同時に記録することができる。 </li><li>セットアップの定期的なモニタリング。 <ol><li>常にそれが無意識であることを確認するために動物を監視します。 注：動物のウィスカーまたは脚の振動復活の指標である。動物の呼吸がぎくしゃくしている場合には、イソフルランの量をわずかに減少させることができる。取得中の強力な組織ドリフトは、マウスの復活の指標である。 </li><li>目的が適切に浸漬していることを確認します。 37℃のPBS betweeリニュー2件の買収（30分）をn個。 注：買収時の信号のプログレッシブ消失は、PBS漏れの指標である。 </li></ol></li></ol></li><li>手続きの終了<ol><li>必要な動画が記録されると、急速に蘇生する前に頚椎脱臼により動物を安楽死させる。 NOTE：創傷治癒は、長手方向の撮像を実行するための可能性を制限24〜48時間でかさぶたの形成をもたらす。 </li></ol></li></ol> 3.データ分析<ol><li>ドリフト補正<ol><li> 3D自動追尾のためのIMARISビットプレーンソフトウェアを使用してください。 </li><li> IMARIS上の3D画像シーケンスを開きます。 SELECTコマンドを「スポット」、そして均一分野で配布可能な場合は、4種類のアンカーポイントの最低すべての時点のマニュアルトラッキング（マウスクリック+シフト）を生成します。 </li><li> 「編集トラック」タブで「正しいドリフト」コマンドを使用して、ドリフト補正を適用します。 </li><li>トンの確認彼のx補正、yおよびzは、特に内の品質は人為的な揺れを回避する。 注：補正が十分でない場合は、他のアンカーポイントを試してみてくださいまたは分析からのビデオを除外する。 </li></ol></li><li>細胞追跡<ol><li>コマンド &quot;新しいスポットを追加」と「トラックスポットの経時&quot;アルゴリズム設定を選択してください。次のステップに進みます。 </li><li> 「ソースチャンネル」コマンドを使用して、関心の全チャネルのいずれかの自動細胞トラッキング手順を適用します。 10μmとしたオブジェクト（すなわち、細胞）の直径。次のステップに進みます。 注：選択は、オブジェクトと測定された信号の特異性の区別に依存します。要するに、全チャネル上の自動追尾が検出された信号が対象物に特異的である場合にのみ可能である。 SHG及びECFPの両方が485ショートパスフィルタの後NDDによって検出されるため、トラッキングは5後NDDによって記録ECFP信号を使用して、より特異的かつ効率的になる00/550バンドパスフィルタ。クラスタ化または高密度のパックされたオブジェクトは、3.1.2のステップに類似した個々のオブジェクトを手動で選択が必要になります。 </li><li> 「品質」フィルタの種類を選択して、非特異的オブジェクトの追跡を除外するようにしきい値を設定。次のステップに進みます。 </li><li> 2スポット間の「最大距離」と「ギャップサイズ」の正確なパラメータを指定して「ブラウンアルゴリズムモーション」を選択してください。トラックの発生を検証します。 </li><li>トラックが自動的に計算されると、「トラック継続時間」フィルタの種類を選択し、（4時間点を表す）を120秒より短いトラックを排除するためにしきい値を設定。 </li><li> 必須：自動追尾した後、トラックの品質をチェック。 <ol><li>オブジェクトは、トラックパスに従うことを確認するためにタイムバーをアンロール。 「編集」タブで利用可能なトラックポイント補正のオプションを使用しない場合は、次の「削除」コマンド（任意のトラックpを削除して虚偽または不適切なトラックを削除個別に「切断」コマンド）を使用してOINT。トラックパスが不足している時点（マウスクリック+シフト）を追加し、不連続である場合には、所定のトラック上のすべての点を選択し、「編集トラック」タブでコマンドを「接続」を使用します。 </li></ol></li></ol></li><li>定量パラメータ<ol><li> 「環境設定」コマンドで「統計」タブを選択し、リスト内の関心の動的パラメータを選択します。選択してコマンドおよび生成されたExcelファイルを保存」ファイルにすべての統計情報をエクスポート」。 注：いくつかの動的なパラメータを直接、トラックの長さ、瞬間速度、平均速度などのソフトウェアから得られ、真直度を追跡している。運動係数6、20を決定することができるが、ソフトウエアにより直接提供されない。 </li><li>各セルについて、時間（ΔT）の各期間の変位の平均値を算出（30秒タイムラプス映画のために、ΔTは30、60、90秒、などとなります）。時間間隔の関数としての平均二乗変位をプロットする。曲線の直線部分の傾きを計算することによって、運動性係数を得る。 </li></ol></li></ol>

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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=In+vivo+imaging+of+specialized+bone+marrow+endothelial+microdomains+for+tumour+engraftment.">In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment.</a> Nature. 435, 969-973 (2005).</li><li>Cariappa, 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=Perisinusoidal+B+cells+in+the+bone+marrow+participate+in+T-independent+responses+to+blood-borne+microbes.">Perisinusoidal B cells in the bone marrow participate in T-independent responses to blood-borne microbes.</a> Immunity. 23, 397-407 (2005).</li><li>Junt, T., 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=Dynamic+visualization+of+thrombopoiesis+within+bone+marrow.">Dynamic visualization of thrombopoiesis within bone marrow.</a> Science. 317, 1767-1770 (2007).</li><li>Zoumi, A., Yeh, A., Tromberg, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cells+and+extracellular+matrix+in+vivo+by+using+second-harmonic+generation+and+two-photon+excited+fluorescence.">Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence.</a> Proc Natl Acad Sci U S A. 99, 11014-11019 (2002).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1873 (2002).</li><li>Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choreography+of+cell+motility+and+interaction+dynamics+imaged+by+two-photon+microscopy+in+lymphoid+organs.">Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs.</a> Annu Rev Immunol. 26, 585-626 (2008).</li><li>Yost, C. 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=Impaired+neutrophil+extracellular+trap+(NET)+formation:+a+novel+innate+immune+deficiency+of+human+neonates.">Impaired neutrophil extracellular trap (NET) formation: a novel innate immune deficiency of human neonates.</a> Blood. 113, 6419-6427 (2009).</li><li>Devi, 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=Multiphoton+imaging+reveals+a+new+leukocyte+recruitment+paradigm+in+the+glomerulus.">Multiphoton imaging reveals a new leukocyte recruitment paradigm in the glomerulus.</a> Nat Med. 19, 107-112 (2013).</li></ol>

The authors have nothing to disclose.

インビボイメージング方法の重要な点は、画像の持続時間を最大にし、炎症細胞の動態に影響を与える可能性のある細菌汚染および炎症の危険性を最小限にするために、焦点の安定性を確保するためである。骨髄へのアクセスを得るために行わ手術が最小であるように、頭蓋骨骨​​髄のイメージングは​​、これらの目的に従う。滅菌材料および防腐剤の使用は、細胞恒常性?...

The bone marrow plays a central role in hematopoiesis and represents the main reservoir of monocytes that constitutively recirculate between the blood and the medullar parenchyma, renew the pool of circulating monocytes with a short life span 2,3 and participate in the reconstitution of the steady state tissue-macrophages and dendritic cells 4. During inflammation or after transient aplasia, monocytes are actively mobilized from either the bone marrow or the spleen 5, 6, 7 and colonize inflamed organs. Several chemoattractant axis have been involved in the process of myeloid cell mobilization from the bone marrow 8, 5, 6,9. Beyond the myeloid compartment the bone marrow is also an important site of T lymphocyte priming 10 and a niche of immunological memory 11,12. Thus, this tissue is central for numerous investigations in the field of hematology and immunology. Our knowledge on the structural organization of medullar myeloid cells mainly arises from the analysis of histological section of fixed tissues 13. This static view does not allow for a study of the cellular exchange dynamic between the different compartments of the bone marrow, which is the basis of its functional activity.
Intravital imaging constitutes an important biological input in the study of cell mobility, cell adherence and cell-to-cell interactions, which were previously described only from in vitro systems. Technical challenges for proper intravital imaging include the ability to reach the tissue of interest in an optical point of view, and to maximize its isolation from physiological (breath, muscle or peristaltic contractions) or mechanical drifts (tissue disruption and extension following surgery, and exposure to microscope objective as well as temperature and vascular/oxygenation perturbations). Microscopic drifts may limit the ability to keep the focus a long time and could introduce artifacts in the quantification of cell motility. One alternative, validated for several tissues to reduce these technical difficulties, is to work on explanted tissue incubated in a thermostated and oxygenated medium; however, complete disruption of the lymphatic and vascular circulation may be problematic. Intravital imaging of skull bone marrow has several advantages concerning these issues. Firstly, it requires minimal surgical action. Secondly, thickness of the bone in this region allows direct visualization of bone marrow niches without abrasion, thus reducing physiological perturbations. The medullar network can be imaged in the parasagittal region of the bone; however the sinusoids are more visible in the fronto-parietal area where the bone matrix is thinner12,14. 
Intravital imaging relies on the availability of the most accurate fluorescent reporter tagging the population of interest. In vitro labelling of purified cell population before adoptive transfer led to important characterization of hematopoietic stem cell niches 15 or bone marrow endothelial microdomains favouring tumor engraftment 16, and provided several fundamental inputs on key concepts in immunology 17 . However, this approach usually requires hundreds of thousands of cells to get a chance to detect them afterwards in vivo. This could be explained by the high mortality rate following staining, the dilution in the whole body and the change in the activation state, which might lead to biased homing. Endogenous tagging from transgenic mouse system greatly overcomes these limitations and has allowed to image the behaviour of endogenous osteoblast 8, megakaryocytes 18 or myeloid-lineage subsets 6 . Nevertheless, one has to be cautious when considering the specificity of the fluorescent reporter among the studied subset. 
The Csf1r-Gal4VP16/UAS-ECFP, called MacBlue mouse 1, is a valuable transgenic system to study medullar monocytes with real time imaging 6. Intravenous injection of high molecular weight rhodamin-dextran distinguishes the medullar parenchyma from the vascular sinusoid network of the bone marrow. Using this approach, it is possible to track the monocyte behaviour in the different medullar compartments in a specific physiopathological context of interest. Furthermore, we propose an additional strategy to compare monocyte dynamics with that of neutrophils through in vivo labelling using a specific antibody.

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			<td style="width:171px;">Queryo Medical</td>
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			<td>Surgical Glue, rubber ring fixation</td>
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			<td>Carl Zeiss</td>
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			<td height="21" style="height:21px;">Ti:Sapphire crystal laser&#160;</td>
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			<td height="21" style="height:21px;">Zen 2010</td>
			<td>Carl Zeiss</td>
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			<td>Analysis Software, 3D automatic tracking</td>
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			<td>Carl Zeiss</td>
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			<td>intravital imaging</td>
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tracking mouse bone marrow monocytes in vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

マウスの頭蓋骨構造は、生体内イメージングによって骨髄生理学を勉強する良い機会を提供しています。フロント頭頂領域の周りに薄くなる骨は、それが骨の摩耗なし髄ニッチへのアクセスを得ることが可能である。1 MacBlueトランスジェニックマウスの頭蓋骨の広い2Dフィールドを表す図 。骨基質は、主にSHG 19によってIは容易に検出可能なコラーゲンで構成...

Watch this Scientific Journal Video about Tracking Mouse Bone Marrow Monocytes In Vivo at JoVE.com

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

マウス骨髄単球の追跡<em&gt;インビボ</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

tracking-mouse-bone-marrow-monocytes-in-vivo

Research

JoVE Journal

Immunology and Infection

Tracking Mouse Bone Marrow Monocytes In Vivo

9.4K ビュー.  Sorbonne Universités, UPMC Univ Paris 06, CR7. Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism. 

ビデオ: Tracking Mouse Bone Marrow Monocytes In Vivo

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

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

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

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

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

免疫・感染学

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

骨髄移植によるマウス大腸炎における免疫および非免疫細胞からの遺伝子発現の機能的役割を差別化

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

機能研究のためのマウス骨髄好中球と養子移入実験の分離、精製とラベリング

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

骨髄由来マクロファージの生産

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

単離とマウス骨髄由来の単球の静脈注射

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

マウス後肢長骨解剖や骨髄の単離

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

以下のための大腿骨のウィンドウチャンバーモデル<em&gt;インビボ</emマウス骨髄内&gt;細胞トラッキング

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

マウスの骨髄から形質細胞様樹状細胞の精製のために、蛍光活性化細胞選別

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

寡能性と系列拘束されたマウスの骨髄から骨髄前駆細胞の分離と同定

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

An &lt;em&gt;In Vivo&lt;/em&gt; Mouse Model to Measure Na&amp;#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

メジャー ナイーブ cd4 陽性 T 細胞の活性化、増殖と骨髄由来樹状細胞による th1 細胞分化をIn Vivoマウス モデル

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...