この作品は、CNRS（PICS EGに2012年から2014年）によって、およびRegioneカンパニア州（のLR N.5、ジョヴァンナモットラに2002年3月28日）からの助成金によってサポートされていました。フィリッポ·コンティは、科学的な協力財団&#39;&#39; Infectiopoleシュッの仲間である。&#39;&#39;ニコラBoucheritが研究技術のためのフランスの省の仲間です。資金源は、研究デザイン、データ収集、データ分析、公開することを決定、または原稿の準備に何の役割がありませんでした。

倫理に関する声明規則に合うエクスマルセイユ大学の動物取扱プロトコル私たちの施設内動物倫理委員会によって承認された「Conseilの科学研究·デュ·センタード·形成エトデルシェルシュ実験メディコ - 外科的」（エリック·GhigoにCFREMC、プロジェクト許可証10から300122013）のDécretNは1987年10月19日の87から848の°。実験はFacultéデMEDECI​​NE·デ·ラ·ティモーネ（エリックGhigoの実験の許可番号13.385）で実施した。 1。材料および培養培地の準備<ol><li> 2鉗子、2はさみ、2外科ブレード、乳鉢と乳棒を殺菌。 </li><li> 10％ウシ胎児血清（FCS）を含有する完全DMEMを得、2 mMグルタミン、100 U / mlペニシリンおよび100μg/ mlのストレプトマイシン。 </li><li> 1X PBSを取得するために、滅菌蒸留水で10倍のPBSを希釈。 </li><li>氷のように冷たい1X PBSで、氷のように冷たい完全DMEM、完全DMEM WAを取得37℃にrmed </li></ol> 2。 L929細胞上清の調製<ol><li> 37℃、5％CO 2で完全DMEM中で密集するL929細胞（20 175cm 2のフラスコ）成長する。 </li></ol>注：因子（GM-CSF）を刺激する顆粒球-マクロファージコロニーは、マクロファージ12に造血細胞の分化を誘導するために必要とされる。 L929細胞は、GM-CSFを生産する。 <ol start="2"><li>合流点に、新しい完全DMEMで培養培地を交換してください。 10日間、32℃、5％のCO 2へのフラスコを転送します。 </li><li>収集、プールと10分間750×gで上清を遠心する。細胞ペレットを廃棄する。 </li><li> -20℃で15 mlチューブや店舗内の店舗上清</li></ol> 3。骨髄由来マクロファージ（BMDM）の準備<ol><li>頚椎脱臼により1マウスを生け贄に捧げる。 <ol><li>実験を通して無菌外科刃を使用してください。 70％のALCOで皮膚を消毒HOL。各後肢の上部に切開を行い、筋肉を露出させ、足の方に皮膚をプルダウン。 </li><li>後ろ足を切断し、滅菌したはさみや滅菌ピンセットで皮膚を取り除きます。無菌の氷冷した1×PBS（5ミリリットル）を含む滅菌ペトリ皿（35/10ミリメートル）以内に足を置きます。 </li><li>無菌はさみやピンセットで骨に付着している肉と筋肉を外します。 </li></ol></li><li>氷のように冷たい、無菌1X PBS（5ミリリットル）を含む新しい滅菌ペトリ皿（35/10ミリメートル）に骨を移す。 5ミリリットルの氷冷滅菌1×PBSで骨を2回洗浄する。 </li><li> 5ミリリットルの氷冷滅菌1X PBSを含む無菌乳鉢に骨を移す。 </li><li>無菌ハサミとの共同での大腿骨から脛骨を切った。乳棒を用いて5ミリリットルの氷冷滅菌1X PBSを含む無菌乳鉢で静かに骨を粉砕。 </li><li>氷のように冷たい15ミリリットルチューブに上清を収集します。このステップ3回繰り返します。 </li><li> 70μmのナイロン細胞STRAでろ過固体断片を除去するためにINER。 4℃で10分間450×gでろ液を遠心分離</li><li>そっと上澄みを捨てる。 10mlの30秒間の赤血球溶解緩衝液をペレットに解離する。 20ミリリットルの氷のように冷たい、完全DMEMを追加します。 </li></ol>注：このステップの目的は、赤血球を汚染除去することであるため、このステップは、赤血球溶解緩衝液によって造血細胞変質を避けるために、2分以内に行わなければならない。 <ol start="8"><li> 4℃で10分間450×gで遠心分離そっと上澄みを捨てる。 37℃に温められた20ミリリットル完全DMEMにペレットを解離</li><li> 2ペトリ皿（100/20ミリメートル）に解離細胞を移す。 37℃で4時間、それらをインキュベート</li><li>室温で50 mlチューブに上清を収集します。常在性マクロファージが含まれている料理を捨てる。 </li></ol>注記：このステップの目的は、常駐骨marroを排除することである培養処理プラスチックに付着する能力によってワットマクロファージ。これらの常在マクロファージは、他の実験において使用され得る。 <ol start="11"><li> 4℃で10分間450×gで回収した上清を遠心分離上清を捨てる。 </li><li> 10ミリリットル15パーセントL929細胞上清を含む完全DMEM中で静かにペレットを解離。 40μmのナイロン細胞濾過器細胞をフィルタリングします。 </li><li>ろ液を回復します。 140ミリリットル15パーセントL929細胞培地を補ってきた完全DMEMに収集されたろ液（10ミリリットル）を追加します。 </li><li>ペトリ皿（15ペトリ皿、20分の100 mm）のあたりの細胞懸濁液10mlに分配する。 37℃、5％CO 2で細胞をインキュベートする。 </li><li> 3日間、細胞を増殖させる</li><li> 10ミリリットル15パーセントL929で補充された完全DMEMを追加します。 4追加日間細胞をインキュベートする。 </li></ol>注：倒立顕微鏡で定期的に細胞増殖をモニターし（ステップ3.14から3.16）。付着性マクロファージはなり培養3日後に観察した。 4。収穫BMDMs <ol><li>上清を取り除きます。完全DMEMで洗浄BMDMs 2回</li><li> 5ミリリットルを37℃に加温された完全DMEMを追加静かにラバーポリスマンでこすることによってBMDMsを外します。 </li><li> 10分間450×gで50ミリリットル試験管や遠心分離器でBMDMsを収集します。静かに20ミリリットルの完全DMEM中で細胞ペレットを解離する。 </li><li>パンブルーの存在下でBMDMsを数える（10％以下の死亡率が観察されないはずである）。 </li></ol>注意：一般に、約6〜7.5×10 7のマクロファージが、マクロファージの15ペトリ皿（100/20ミリメートル）から得られる。約4〜5×10 6のマクロファージ/シャーレ（100/20ミリメートル）があります。 <ol start="5"><li>実験に必要とされるBMDMsを準備します。 37℃で完全培地で16時間後、5％のCO 2が 、マクロファージが再び支持体に付着する。 </li></ol> 5。 BMDMsを保存する<ol><li>孤立BMDMs（ステップ4.3）を収集。 10分間450×gで遠心分離する。注：BMDMsを液体窒素中で凍結されてもよい。 </li><li>各アンプルに4×10 6細胞/ mlピペット1mlを、最終濃度10％DMSOおよび90％FCSからなる凍結培地に再懸濁した細胞ペレット。 </li><li> 1℃/分の冷却速度で細胞を凍結する。 24時間後、長期保存のために液体窒素容器をアンプルに移す。 </li></ol>

<ol>
	<li>Rutherford, M. S., Witsell, A., Schook, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+generating+functionally+heterogeneous+macrophages:+chaos+revisited.">Mechanisms generating functionally heterogeneous macrophages: chaos revisited.</a> J. Leukocyte Biol. 53, 602-618 (1993).</li><li>Van Furth, R., Gallin, J. I., Goldstein, I. M., Snyderman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Inflammation: Basic Principles and Clinical Correlates. 112, 325-336 (1992).</li><li>Adams, D., Halmiton, T., Gallin, J. I., Goldstein, I. M., Snyderman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Inflammation: Basic Principles and Clinical Correlates. 112, 325-336 (1992).</li><li>Morris, L., Graham, C. F., Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+in+haemopoietic+and+other+tissues+of+the+developing+mouse+detected+by+the+monoclonal+antibody+F4/80.">Macrophages in haemopoietic and other tissues of the developing mouse detected by the monoclonal antibody F4/80.</a> Development. 112, 517-526 (1991).</li><li>Haas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phagosome:+compartment+with+a+license+to+kill.">The phagosome: compartment with a license to kill.</a> Traffic. 8, 311-330 (2007).</li><li>Underhill, D. M., Ozinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+of+microbes:+complexity+in+action.">Phagocytosis of microbes: complexity in action.</a> Annu. Rev. Immunol. 20, 825-852 (2002).</li><li>Castagna, A., Polati, R., Bossi, A. M., Girelli, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte/macrophage+proteomics:+recent+findings+and+biomedical+applications.">Monocyte/macrophage proteomics: recent findings and biomedical applications.</a> Expert Rev. Proteomics. 9, 201-215 (2012).</li><li>Benoit, M., Desnues, B., Mege, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+polarization+in+bacterial+infections.">Macrophage polarization in bacterial infections.</a> J. Immunol. 181, 3733-3739 (2008).</li><li>Barry, A. O., 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+stimulation+of+p38alpha-MAPK/Vps41-HOPS+by+LPS+from+pathogenic+Coxiella+burnetii+prevents+trafficking+to+microbicidal+phagolysosomes.">Impaired stimulation of p38alpha-MAPK/Vps41-HOPS by LPS from pathogenic Coxiella burnetii prevents trafficking to microbicidal phagolysosomes.</a> Cell Host Microbe. 12, 751-763 (2012).</li><li>Henry, R. M., Hoppe, A. D., Joshi, N., Swanson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+uniformity+of+phagosome+maturation+in+macrophages.">The uniformity of phagosome maturation in macrophages.</a> J. Cell Biol. 164, 185-194 (2004).</li><li>Sundaramurthy, V., 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=Integration+of+Chemical+and+RNAi+Multiparametric+Profiles+Identifies+Triggers+of+Intracellular+Mycobacterial+Killing.">Integration of Chemical and RNAi Multiparametric Profiles Identifies Triggers of Intracellular Mycobacterial Killing.</a> Cell Host Microbe. 13, 129-142 (2013).</li><li>Boltz-Nitulescu, G., 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=Differentiation+of+rat+bone+marrow+cells+into+macrophages+under+the+influence+of+mouse+L929+cell+supernatant.">Differentiation of rat bone marrow cells into macrophages under the influence of mouse L929 cell supernatant.</a> J. Leukocyte Biol. 41, 83-91 (1987).</li></ol>

利害の宣言された関係はありません。

ここに説明されたプロトコルは、BMDMsを大量に製造する方法を詳しく説明します。 BMDMは、一次細胞であり、未成熟でマクロファージ細胞株とは対照的に、成熟があるので、生物学的機能及び単球から分化したマクロファージの特性を有する。 BMDMsは、遺伝的スクリーニング（RNAi）と、薬物スクリーニング、機能的研究、宿主 - 病原体相互作用研究および調査の他の多くの分野に使用するこ?...

マクロファージ機能の重要な側面は、自然免疫と獲得免疫におけるその役割です。そのため不活性粒子、細菌や寄生虫を貪食する能力のため、マクロファージは、外国の侵略に対する防御の第一線である。内在化されると、微生物はファゴリソソーム内で分解される。マクロファージはまた、Tリンパ球などの他の免疫細胞に募集のための信号と抗原を提示を送信します。マクロファージは単球に由来している。単球は、骨髄性幹細胞から骨髄で発生し、それらはマクロファージに分化し、末梢血および種々の組織に移動する。これは、健康な成体マウスは、様々な器官および組織に体全体に分布している約10 8マクロファージ（ 表1）1,2 を含有すると推定される。マクロファージは、それらの微小環境の3,4に適応する能力の優れた表現型および機能的多様性を示す。最も重要なmacropヘイグプロパティは、微生物を貪食し、それらを破壊する能力によって定義され、その殺菌活性、である。食細胞応答は、微生物の接触によって刺激される複雑なシグナル伝達ネットワークの活性化によって定義され、したがって、マクロファージは、多様な刺激に応答して、適宜遺伝子発現を調節する。貪食した後、微生物はゴリソソームと呼ばれる構造で排除されますが、多くの病原性微生物はマクロファージ5の殺菌機能を破壊するための戦略を開発した。異なる微生物種によって利用されるSubversionのメカニズムの多様性は、食作用のプロセス6およびファゴリソソーム生合成の複雑さを証明するものです。感染症は、主要なヒトの健康上の問題であり、数多くのメカニズムや分子は、マクロファージの抗菌活動に参加しています。さらに、微生物によってハイジャックされた殺菌特性のターゲットは不明のままであるため、Eがありますマクロファージに関する科学的関心をリニューアルしました宿主 - 病原体相互作用の進化遺伝的および生化学的な側面への関心のXPLOSION。現在、分野の研究の大部分は貪食能、サイトカイン産生と酸化的バーストの調節に初代マクロファージとは異なり、マクロファージ細胞株、上で行われます。さらに、それらは、顕微鏡検査のためにあまり適していない。インタラクション·マクロファージ·病原体を調べるためには、より多くの生理学的な特徴を示すような骨髄由来マクロファージ（BMDMs）などの主要なマクロファージを、使用することをお勧めします。これらのマクロファージは、トランスジェニックマウスから直接単離する可能性があり、例えば、レンチウイルストランスフェクションなどの新しい技術の利用可能性と、それらの遺伝子発現プロフィールが、遺伝子の過剰発現またはRNA干渉によって変化させることができるので、また、遺伝子改変BMDMs上で動作することができる。ここでは、マウスの骨mを区別するための手順を記載様々な機能のために7日間でマクロファージを多数提供しますマクロファージに矢印は、そのようなプロテオミクス7、トランスクリプトミクス8として分析して、細胞内輸送は9、動的スタディ10、遺伝子スクリーニング（RNAi）とし、薬物スクリーニング11を研究している。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco Life Technologies</td>
			<td>21969-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Gibco Life Technologies</td>
			<td>10270</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco Life Technologies</td>
			<td>15070</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Gibco Life Technologies</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10x)</td>
			<td>Lonza</td>
			<td>BEM515F</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis buffer</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 70 &#956;m Nylon</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 40 &#956;m Nylon</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes (100/20 mm)</td>
			<td>any supplier</td>
			<td>n/a</td>
			<td>culture treated</td>
		</tr>
		<tr>
			<td>Petri dishes (35/10 mm)</td>
			<td>any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

bone marrow-derived macrophage production

マクロファージは自然免疫と獲得免疫応答の重要なコンポーネントであり、彼らは、それらの強力な殺菌活動の外国の侵略者に対する防御の最前線である。マクロファージは体内に広く分布し、リンパ器官、肝臓、肺、胃腸管、中枢神経系、骨、及び皮膚に存在する。ため、その配分のために、それらは、生理学的及び病理学的プロセスの広い範囲に関与している。マクロファージは、微小環境の変化を認識すると、組織の恒常性を維持することができる汎用性の高い細胞である。数々の病原体は、生き残るためにトロイの木馬としてマクロファージを使用中に複製して、人間と動物の両方に感染し、体全体に伝播させるためのメカニズムを進化させてきた。宿主 - 病原体相互作用の進化遺伝的および生化学的な側面に興味の最近の爆発は、マクロファージに関する科学的関心をリニューアルしました。ここでは、説明します宿主 - 病原体相互作用だけでなく、他のプロセスを研究するためにマクロファージを大量に提供するマウス骨髄からマクロファージを分離し、育成するための手順。

この方法の目的は、容易に数日後にマクロファージを大量に得ることであった。骨髄細胞の調製は、 図1に示されている。後肢の骨を収集し、乳鉢で潰した。常在性マクロファージは、骨髄細胞調製物から除去した後、骨髄細胞をGM-CSF（0日目）とインキュベートした。 3日後、培養前のラウンドと非接着であった細胞は、マクロファージに分化し、（ 図1A）を接着す?...

Watch this Scientific Journal Video about Bone Marrow-derived Macrophage Production at JoVE.com

Bone Marrow-derived Macrophage Production

マクロファージは、長い自然免疫と獲得免疫応答の重要な構成要素として認識されている。マクロファージや微生物間の相互作用の進化遺伝的、および生化学的な側面に関する知識の最近の爆発はマクロファージへの科学的関心をリニューアルしました。この記事では、マウスの骨髄からマクロファージを差別化する方法を説明している。

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 ...

bone-marrow-derived-macrophage-production

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JoVE Journal

Immunology and Infection

72.7K ビュー.  Aix-Marseille Université. マクロファージは、長い自然免疫と獲得免疫応答の重要な構成要素として認識されている。マクロファージや微生物間の相互作用の進化遺伝的、および生化学的な側面に関する知識の最近の爆発はマクロファージへの科学的関心をリニューアルしました。この記事では、マウスの骨髄からマクロファージを差別化する方法を説明している。 

ビデオ: Bone Marrow-derived Macrophage Production

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

活性骨髄由来マクロファージとBradyzoite条件におけるトキソプラズマのシスト壁形成

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

免疫・感染学

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 ...

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

骨髄由来マクロファージを用いたマクロファージ分極の調査

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

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 ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

リアルタイム細胞外フラックス分析を用いた偏光M1、M2の骨髄由来マクロファージの代謝特性評価

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

マウス骨髄と腹腔マクロファージ活性化抗体を用いた薬剤効果の評価

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

インフラマソーム活性化とマウス骨髄由来マクロファージにおける Pyroptotic の細胞死の検出

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

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 ...

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

This protocol has been established to culture tetrahydrobiopterin (BH4)- and inducible nitric oxide synthase (iNOS)-deficient primary murine macrophages to study NO-redox biology. The study focuses on reducing potential contamination of BH4 and other artifacts found in traditional isolation and culture methods which may confound experimental outcomes and interpretation of results.

NO-Redox生物学研究のための骨髄由来マクロファージの単離と 体外 培養

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

マウスから骨髄由来樹状細胞を単離および培養するための経済的かつ効率的なプロトコル

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...