この作品は、米国心臓協会（博士Beiyan周にBGIA 7850037）によってサポートされていました。

1。骨髄細胞の単離<ol><li> 6-8週齢のマウスからの大腿骨と脛骨の骨を分離し、髪を洗い流した後、骨を開いてカット。 </li><li>冷PBS +2％の熱不活性化ウシ胎児血清（FBS）（3-5 ML /マウス）に骨髄を洗い流すために21G針とを10mlのシリンジを使用してください。 </li><li>細胞を解離するために21G針を4〜6回を通して骨髄を渡します。 </li><li>細胞塊、骨、毛髪および他の細胞/組織を除去するために70μmのセルストレーナーを通して細胞を渡します。 </li><li> NH 4 Cl溶液（0.8％NH 4 Cl溶液、STEMCELL技術）の3つのボリュームを追加し、赤血球を除去するために10分間氷上でインキュベートする。 </li><li> 4℃で5分間500 xgで細胞をスピンダウン</li><li>冷PBS +2％FBS（20〜50ミリリットル、細胞の量に依存する）で細胞ペレットを再懸濁します。 </li></ol> 2。誘導BMDM形成<ol><li> BMDM成長培地（2×10 6細胞/ mのに孤立した骨髄細胞を再懸濁しL）。 </li></ol> BMDM成長培地： イスコフ改変ダルベッコ培地（IMDM）+ 10％FBS + 15％、濾過（0.2μmの）L-929細胞（ATCC、CCL-1）培養上清（単球コロニー刺激因子、M-CSFを含む）または10 ngの/ mlのM -CSF。 注：L-929細胞上清は、M-CSF 18が含まれています。 5 X 10 5 L-929細胞は6-7日間T75センチ2フラスコに播種され、馴化培地の効果的活動を確保するために、馴化培地は、使用前にフィルター0.45μmの収集と渡されます。培地のアリコートを1〜2ヶ月のためにすぐに使用されるか、または-80°Cに保存することができます。 <ol start="2"><li> 6または12ウェル組織培養プレート（実験デザインに応じて）（コーニングコスター）におけるシード細胞。 </li><li> 3日目に新鮮BMDM増殖培地を変更します。 </li><li> 7日目に、成熟BMDMの形成は、フローサイトメトリー分析を用いて評価されると蛍光体結合抗体の、CD11b及びF4/80を発現する細胞を検出する。 </li></ol> 3。 BMDM偏アクティベ<ol><li> 7日目に、新鮮な刺激培地に変更：M1活性化のために、IMDM、50 ngの/ mlのIFNγと10％FBS、100 ngの/ mlのLPSまたは100 ngの/ mlのLPSを含む使用し、M2活性化のために、と10％FBSを含むIMDMを使用10 ngの/ mlのIL-4および/または10 ngの/ mlのIL-13。 </li><li>暖かい0.05％トリプシンを用いて皿からそれらを取り外すことによって刺激さBMDMsを集め、PBS、10％FBSを含有する細胞を2回洗浄した。 </li></ol> 注：差別、0.05％トリプシン溶液（0.48 mMのEDTA、Invitrogen社を含む）または2-5 mMのEDTAのCaおよびMgを含まないPBSまたはハンクス平衡緩衝液（HBSS）の後に成熟したマクロファージをデタッチし、再懸濁するために使用することができます。トリプシンを含む消化媒体を使用する場合、細胞はシュルの損失を避けるために10分未満、37°C​​で処理する顔はオーバー消化のためにタンパク質。 <ol start="3"><li>標準的なフローサイトメトリー染色手順を使用して、様々な時点でのCD11b、F4/80、CD11cは、CD206、CD69、CD80またはCD86を含む細胞表面抗原の発現を検出する抗体を使用。 </li><li>定量RT-PCRを用いたIL-1β、TNF-αおよびIL-6（M1の活性化）、またはIL-10、IL-13、arginase1およびPPARγ（M2活性化）を含む活性化M1とM2マクロファージの特徴的な遺伝子の発現を決定する。ウエスタンブロット分析によってM1またはM2マクロファージの活性化に関与する細胞のシグナル伝達経路の活性化を決定します。 </li></ol>

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利害の衝突は宣言されていない。

ここでは、骨髄前駆細胞由来のマクロファージの活性化を誘導するためのシンプルかつインビトロで容易に適応手順を報告する。この手順は、マクロファージの偏光を担当するメカニズムの研究のために使用することができる。成熟したマクロファージの純度は、このプロトコルの平均値を95から99までパーセントを用いて得られ、追加の精製法が必要とされない。マクロファージ分極の文脈における利益の特定の遺伝子の機能を調べるために、異所性発現または遺伝子特異的ノックダウンは、7日目に細胞のトランスフェクション後に行うことができる。このプロトコルは、また形成、成熟およびマクロファージの表現型に影響を及ぼす特定の要因や遺伝子の影響を調査するために7日間培養ウィンドウを提供します。 活性化マクロファージは、様々な刺激3、9、19に対応して大きな可塑性を持つ複雑な細胞および分子のプロファイルを表示します。のためにしかしながら、活性化プロファイルが完全に重なっていない、例えば、LPSは、IFNγまたは腫瘍壊死因子（TNF）およびIL-4およびIL-13 M2活性化を刺激し、両方の存在下でさらに向上させることができる強力な炎症誘発M1応答を誘発する3、4、9、15、19、20。このプロトコルで示された結果は、フローサイトメトリー、定量的RT-PCRとウェスタンブロッティングにより解析した骨髄由来マクロファージと実験の典型的な結果を表しています。表面抗原提示およびマクロファージの活性化に伴う細胞のシグナル伝達経路は、マクロファージ偏1-8広範な文献に記載されて変化する。

古典的な炎症反応とは異なる、組織に浸潤したマクロファージは、多くの場合、宿主組織の生理機能を1-8の調節に重要な役割を果たしている偏光活性状態を表示します。刺激により、マクロファージの活性化は、古典的な（M1）および代替（M2）の活性化2,4,9に分類することができる。 M1マクロファージの活性化は、例えばTNF-αとし、炎症性サイトカインの産生をもたらす、Toll様受容体（TLR）および核因子カッパB（NFκB）/ c-Jun N末端キナーゼ1（JNK1）の活性化に依存するIL- 1β、例えば、窒化酸化物（NO）10、11などの反応性酸素種の産生増加をもたらすことのiNOSの活性化。対照的に、M2マクロファージ活性化リクルートPPARγ、PPARδ、またはIL-4-STAT6経路は、代替マンノース受容体CD206のアップレギュレーションと関連付けられ、抗炎症性（M2）活性化、およびアルギナーゼ1（Arg1の）6,12につながる- 14 &lt;/&gt;（商標）。 骨髄由来マクロファージ（BMDM）は活性化マクロファージ15の偏光を制御するメカニズムを理解するためのin vitroモデル理想を提示する。 M2マクロファージの偏光IL-4および/またはIL-13によって誘導することができるながら具体的には、M1マクロファージの活性化は、リポ多糖（LPS）刺激によって誘導することができる。成熟した骨髄由来マクロファージおよび活性化マクロファージがCD11bを、F4/80、CD11cは、CD206、CD69、CD80およびCD86 9、16、17を含む表面抗原の発現についてフローサイトメトリー分析によって同定することができる。また、マクロファージ偏光に関連付けられたシグナル伝達経路のサイトカイン産生および細胞の変化は、それぞれ、定量的RT-PCRおよびウエスタンブロットによって測定することができる。要約すると、マウスの骨髄由来マクロファージは、in vitroでマクロファージ分極を研究するため、関連するモデルとしての役割を果たすことができます。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>IMDM</td>
 <td>Thermo Scientific</td>
 <td>SH30259.01</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Invitrogen</td>
 <td>10438-026</td>
 <td></td>
 </tr>
 <tr>
 <td>Murine GM-CSF</td>
 <td>PeproTech</td>
 <td> 315-03</td>
 <td></td>
 </tr>
 <tr>
 <td>NH4Cl</td>
 <td>StemCell Technologies</td>
 <td>7850</td>
 <td></td>
 </tr>
 <tr>
 <td>L-929</td>
 <td>ATCC</td>
 <td>CCL-1</td>
 <td></td>
 </tr>
 <tr>
 <td>70 &mu;m cell strainer</td>
 <td>BD Biosciences</td>
 <td>352350</td>
 <td></td>
 </tr>
 <tr>
 <td>10 x PBS</td>
 <td>Thermo Scientific</td>
 <td>AP-9009-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse CD11b-APC</td>
 <td>eBioscience</td>
 <td>17-0112-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse F4/80-FITC</td>
 <td>eBioscience</td>
 <td>11-4801-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse CD69-PE</td>
 <td>eBioscience</td>
 <td>12-0691-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse CD86-PE</td>
 <td>eBioscience</td>
 <td>12-0862-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Propidium Iodine</td>
 <td>Invitrogen</td>
 <td>P3566</td>
 <td></td>
 </tr>
 </tbody>
</table>

investigation of macrophage polarization using bone marrow derived macrophages

記事では、マクロファージ分極を調査するためのin vitroモデルで容易に簡単に適応を説明しています。 GM-CSF/M-CSFの存在下で、骨髄からの造血幹/前駆細胞は、M1またはM2の刺激に続いて、単球分化に向けられる。活性化状態は、細胞表面抗原、遺伝子発現および細胞シグナル伝達経路の変化によって追跡することができる。

BMDM生成手順の概略的な説明は、（ 図1）提示される。彼らはCD11bを+ F4/80 +細胞の95〜99％（ 図2）を表す場合、成熟マクロファージの高純度は、7日目に観察することができる。偏マクロファージは、フローサイトメトリー分析を行ったCD11b、F4/80、CD11c陽性およびCD206に対する抗体を用いて調べることができる。 図3に示すように、M1マクロファージとして検出されたCD11b + F4/80 +のCD11c + CD206細胞（Q2）、M2マクロファージのCD11b +であるF4/80 +のCD11c-CD206 +細胞（Q4）。 図に示されているように：BMDMアクティベーションステータスが増加し、細胞サイズ（ 図4A、FSCイン右シフト）およびマクロファージの表面抗原CD69、CD80、またはCD86の増加豊富（のCD11b + F4/80 +門）で確認することができる4B。サイトカイン産生、遺伝子発現およびM1またはM2マクロファージ細胞シグナル伝達経路の活性化は、定量的RT-PCRまたはウェスタンブロッティングを用いて評価することができる。 Asは、図5に示すように、アルギナーゼ1（引数1）及びPPARγレベルは、10 ngの/ mlのIL-4刺激によりM2マクロファージに増加した、IL-1βおよびTNFα産生を100 ngの/ mlのLPSで刺激されたマクロファージ（M1）に増加したのに対し、p65の活性化（ 図6）が付属している。 <img src="/files/ftp_upload/50323/50323fig1.jpg" alt="図1" fo:content-width="5.5in"/> 図1。分離、形成、マウスBMDMsの刺激のためのスキーム。ステップ手順バイステップは、テキストで説明されています。要するに、大腿骨と脛骨の骨はPBSを用いて洗い流し6-8週マウスおよび骨髄細胞から収集され、2％熱不活性化FBSを補充した。赤血球を塩化アンモニウム溶液で溶解した後、細胞を細胞集団の純度成熟および分析を行っ7日間BMDM増殖培地で培養する。マクロファージ経口の分析のため<html> larization、細胞を活性化M2 +IFNγM1、又はIL-4および/またはIL-13に対してLPS又はLPSで刺激した。偏マクロファージ細胞形態の変化、表面マーカープレゼンテーション、サイトカイン産生および活性化細胞シグナル伝達経路に基づいて評価することができる。 <img src="/files/ftp_upload/50323/50323fig2.jpg" alt="図2" fo:content-width="6in" fo:src="/files/ftp_upload/50323/50323fig2highres.jpg"/> 図2。流れBMDM形成のサイトメトリー分析。（）BMDMsは破片と結合体を除去するためにFSCとSSCの最初のゲーティングされた。（B）熟BMDMsがのパーセンテージとして表示純度のCD11b + F4/80 +亜集団（右上）のように定義されたFSC / SSCにゲート母集団。 s/ftp_upload/50323/50323fig3highres.jpg &quot;/&gt; 図3。 。マクロファージ偏光解析組織浸潤マクロファージのCD11b + F4/80 +細胞として定義され、M2マクロファージのCD11b + F4/80 +のCD11c - CD206 +細胞であるのに対し、M1マクロファージは、のCD11b + F4/80 +のCD11c + CD206-細胞である。 <img src="/files/ftp_upload/50323/50323fig4.jpg" alt="図4" fo:content-width="6in" fo:src="/files/ftp_upload/50323/50323fig4highres.jpg"/> 図4。フローサイトメトリーによるマクロファージ活性化分析。（）活性化されると、マクロファージのサイズは、M0（未処理）マクロファージに比べ刺激後24時間におけるM1またはM2マクロファージのFSC-のシフトによって証明増やします。（B）活性化関連表面マーカーは、IL4又はLPS刺激後のフローサイトメトリーにより分析した。早い応答マーカーCD69 5または24時間刺激後に評価された; CD80およびCD86マルケRSは刺激後48時間で分析した。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50323/50323fig4large.jpg" target="_blank">より大きい数字を表示するには、ここをクリックしてください</a> 。 <img src="/files/ftp_upload/50323/50323fig5.jpg" alt="図5"/> 図5。 24時間10 ngの/ mlのIL-4で刺激代替マクロファージの活性。マクロファージを収集し、全RNAを抽出した。定量的RT-PCR解析を用いて未処理のマクロファージと比較して上昇したアルギナーゼ1（引数1）及びPPARγ発現はM2マクロファージで検出された。 <img src="/files/ftp_upload/50323/50323fig6.jpg" alt="図6" fo:content-width="5.5in" fo:src="/files/ftp_upload/50323/50323fig6highres.jpg"/> 図6。マクロファージの古典的な活性化、（A）全RNAを、100 ngの/ mlのLで刺激されたマクロファージから抽出された定量的RT-PCR分析で24時間、使用のためにPS。 IL-1βおよびTNFαを含む炎症性サイトカインの発現は、M1マクロファージにおいて増加した。P65に対する抗体を用いて決定し、ウェスタンブロット分析でのp65のリン酸化と（B）NFκB経路の活性化は、LPSにより誘導した。

Watch this Scientific Journal Video about Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages at JoVE.com

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

記事は容易に簡単に適応を説明<em&gt; in vitroで</emマクロファージ分極を調査する&gt;モデル。 GM-CSF/M-CSFの存在下で、骨髄からの造血幹/前駆細胞は、M1またはM2の刺激に続いて、単球分化に向けられる。活性化状態は、細胞表面抗原、遺伝子発現および細胞シグナル伝達経路の変化によって追跡することができる。

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 ...

investigation-macrophage-polarization-using-bone-marrow-derived

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

65.9K ビュー.  Texas A&M University. 記事は容易に簡単に適応を説明 in vitroでモデル。 GM-CSF/M-CSFの存在下で、骨髄からの造血幹/前駆細胞は、M1またはM2の刺激に続いて、単球分化に向けられる。活性化状態は、細胞表面抗原、遺伝子発現および細胞シグナル伝達経路の変化によって追跡することができる。

ビデオ: Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

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 ...

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 ...

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 ...

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, ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

マウスの骨髄からGM-CSF由来肺胞のようなマクロファージや樹状細胞の生成および同定

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

マクロファージコロニー刺激因子分化したヒト単球由来マクロファージの文化

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

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.