This work was supported by the DFG (Deutsche Forschungsgemeinschaft, German Research Foundation) SFB 854 (Sonderforschungsbereich, collaborative research center). 
Thanks to Hans-Holger G&#228;rtner, Audiovisuelles Medienzentrum, Otto-von-Guericke University Magdeburg, Magdeburg, Germany, for technical support.

この研究は、動物保護（24D-9168.11から1/2008から24）のためのドイツ法第8に従って、ザクセンとザクセン=アンハルト、Regierungspraesidiumドレスデン/ハレの国の許可を得て実施した。 1細胞の単離 1.1大腿骨と脛骨の調製を<ol><li>閉じたビンにイソフルランを気化させることにより、5％イソフルラン濃度を用いて、マウスを麻酔。マウスは3秒間移動を停止した後、頚椎脱臼を行う。 </li><li>エタノール（96％）で後肢を消毒する。皮膚や筋肉を取り出し、無菌のメスで骨を分離する。 <ol><li>鼠径皮膚切開を開始し、内果に向かって切開を延長し、下の子牛の周りの皮膚の円形切開を行う。近位にそれを剥離することにより、完全に足の皮膚を削除します。 </li><li>鋭いメスで大腿骨から大腿四頭筋を切り離し、前方腓骨の両方を削除と腓腹筋グループと靭帯を探し、解剖によって股関節で足をdisarticulate。 </li><li>前方に起動して、慎重に足を回転させ、靭帯を横に切開することにより、関節を中心に進んで、それによってジョイントをdisarticulating。 </li></ol></li><li>洗浄大腿無菌細胞調製を保証する90秒以上96％エタノールで脛骨。残りのエタノールを洗い流すために滅菌PBSで洗浄プロセスを続行します。 注：すべてのその後の工程は、汚染を避けるために無菌でなければならない。 </li></ol>骨髄の1.2収穫<ol><li>大腿骨と脛骨のシャフトへのアクセスを得るために細かいハサミで各骨の近位端および遠位端をカットします。これらの骨は非常に簡単に壊れているため、このステップには注意してください。 </li><li>温かい培地で骨をフラッシュする（M199 + 10％ウシ胎児血清（FCS）+ 1％ペニシリン/ストレプトマイシン）。無菌の28-G針、1ミリリットルの注射器、10〜15 mlの培地あたりの間に使用しますすすぎのための骨。 </li><li>細かい鉗子で骨を持ち、それが半透明に変わるまで一つ一つをすすぐ。注意深く細胞ストレスを最小にするために、シリンジプランジャの端部に圧力を加える。 </li><li> 70μmのナイロンウェブを通じて骨髄を濾過し、ろ液を集める。骨髄の最大量を抽出するために、遠位端と近位端から繰り返しすすぐ。 </li><li> PBSでふるいをすすぎ、ろ液を集める。慎重に穏やかに撹拌し、ピペッティングにより破片や携帯コングロマリットを取り除く。 </li></ol> 1.3栽培<ol><li> RTで10分間250×gで細胞懸濁液を遠心します。上清を捨て、約25ミリリットルの培地で細胞を懸濁します。一回繰り返します。 </li><li>第2洗浄工程の後に培地6mlの中に細胞を再懸濁する。パンブルーの50μlの細胞溶液50μlを混ぜる。光学顕微鏡下で計数チャンバー内の細胞を数える。数cを計算するこの式を使用して、溶液中のエル： <img alt="式1" fo:content-width="1.3in" src="/files/ftp_upload/52347/52347eq1.jpg" /></li><li>プレートの底に永久的な付着を防止するために、6ウェルの超低取付面板上に細胞を播種する。ウェルあたり最大6 mlの1mlあたり10 6細胞の濃度を使用してください。 </li><li>細胞分化を促進するために20ng / mlのM-CSFとの懸濁液を補う。 </li><li>培養物を37℃で5日間、細胞を、5％の二酸化炭素とを毎日観察する。 </li></ol>細胞の1.4収穫注：これらの手順は、氷上で行われるべきである。それに応じて1.4.1または1.4.2のどちらかに進みます。 <ol><li>この時点で、彼らは、超低取付面板であっても、培養プレートに付着する分化したマクロファージを含む、全体の培養を収穫する。 <ol><li>穏やかなピペッティングにより全体の文化を収穫し、4℃、PBの溶液で脱着S、0.5％ウシ血清アルブミン（BSA）および2mM EDTA。わからない場合には、顕微鏡下で確認する - プレートが接着細胞を残りの明確になるまで繰り返します。 </li></ol></li><li>別の方法として、主に単球が含まれている懸濁液中の細胞と上清を収穫することにより、接着性マクロファージを捨てる。 <ol><li> EDTA非含有PBS溶液と0.5％BSAと非接着細胞を回収。穏やかに付着したマクロファージを追い出し避けるためにあまりにも多くの力を加えないでください。 </li></ol></li></ol> 1.5 FACS-解析（オプション） 注：これは、MACSを用いてCD117 +ステムおよび前駆細胞の細胞懸濁液を枯渇させることが可能である。この手順については、製造元のプロトコルを使用してください。 <ol><li>どちらかが1.4.1または1.4.2の手順に従って、細胞を採取する。 </li><li> 4℃で10分間250×gで細胞を遠心して、もう一度この手順を繰り返します。 </li><li> FACS bの300μlの中に再懸濁し、少なくとも250,000細胞プローブあたりuffer。 </li><li> 96ウェルプレート（ウェルあたり30μL）の上に細胞溶液を転送します。 </li><li> 4℃で5分間250×gで細胞懸濁液を遠心します。 </li><li>上清を除去し、各ウェル（使用メーカー希釈）に対する抗体の25μlを添加する。 </li><li>抗体と細胞（1.5.1-1.5.6ステップ後の細胞表現型のために製造業者のプロトコルを以下のように染色。単球/マクロファージの同定のために一般に使用されるマーカーは、CD11bの、F4 / 80（ 図3）、CD115（図2）、およびGR-を含む1。 注：セル·ソリューションのさらなる説明については、CD4、CD8a、のCD11c、CD25、CD45、CD45R、CD62L、CD80、CD86、CD145、CD117、MHCⅡ、Ly6C、Ly6G、およびCD192のようなマーカーの使用が推奨されます。細胞の特徴は、以前に12を説明した。 </li><li> 4℃で20分間、プローブをインキュベートする。 </li><li>遠心機で4℃で5分間250×gでプローブし、FACS緩衝液200μlに再懸濁とは目を繰り返しますステップの2倍である。 </li><li> FACSチューブにプローブを転送し、FACS分析12を行う。 </li><li>細胞分化を分析するために、0日目、3及び5にFACS分析12を実行する。 </li></ol> 2.尾静脈注射 2.1準備<ol><li>約10分間37℃で加熱板上で動物を暖める。過熱の兆候を認識するために温めながら、動物を観察します。 </li><li> NaClを150μlの中で、ステップ1.1〜1.4で孤立し、単球を再懸濁し、30Gの針を1ミリリットルのインスリン注射器に細胞をロードします。軽くすべての球が注入されるように、注射器をロードする前に解決策をボルテックス。常に熱媒介アタッチメントと単球の活性化を避けるために、氷上で細胞を処理します。 </li></ol> 2.2保全処分<ol><li>静脈内注射のためのマウスを選択する際にケージ内の他のマウスを乱すことは避けてください。 </li><li>制止にマウスを置きます。あまりにも多くのPRを避けるessureと後肢に注意してください。 （ 図4） 注：または、動物のストレスのないハンドリングを保証するために全身麻酔を使用しています。 </li><li>マウスが制止を閉じる前に、呼吸のためのスペースがあることを確認してください。 </li></ol> 2.3インジェクション<ol><li>消毒剤と、注射部位を消毒する。マウスの尾にスプレーし、少なくとも1分間放置。 </li><li>静脈の視認性のために、注射部位の上に外側尾側に緩やかな圧力を加えることにより、尾の静脈血の流れを止める。 </li><li>注射の前に、尾を90度回します。 （ 図5） </li><li> 45度の角度で注入する。動物に害を避けるために、ゆっくりと何グラムあたり5μL以上の注入ません。 </li><li>失敗した注射のサインですブリスターがある場合は、すぐに中止し、より近位に注射を繰り返します。 </li><li>穏やかなページを適用することにより、射出側の出血を止める約1分間の案内圧力スイッチ。 </li><li>手順の最後に、リトラクターを開き、そのケージにマウスを置く。 </li><li>動物が注射によって害されませんでしたし、胸骨の横臥が維持されることを保証するために20分間、マウスを観察します。マウスが十分な意識を取り戻すまで、観測の時間を延長します。それは完全に回復した後にのみ、他の動物の会社にマウスを返します。 </li></ol>

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The authors declare that they have no competing financial interests.

我々は、骨髄由来のマウスの単球を大量に単離するための単純で費用効果的な方法を記載する。 1.4×10 6の単球収率5を得た末梢血を使用して、他のプロトコルと比較して、我々は、単一のドナーマウスから11×10 6±3×10 6単球のより高い収率を得ることができる。 この方法の課題を考慮した場合、非無菌条件下で作業する場合には、汚染の可能性を言及することが重要である。すすぎ後の洗浄工程が適切に従わない場合は、細菌および真菌による汚染可能性が高いです。さらに、骨髄は顆粒球、リンパ球および赤血球系細胞を含む不均一な細胞懸濁液、で構成されています。通常、これらの細胞は、増殖因子の欠如のために2日培養の3との間に消える。成長因子駆動型分化した後、マクロファージは、汚染の主な原因である。トンを維持するためにマクロファージへの単球の分化を最小限に抑えるために、低およびマクロファージの彼数は、超低付着性プレートと、上述した洗浄工程の使用が重要である。 これは、排他的に単球に特異的であるマーカーを同定することは困難である。マーカー間の干渉は、複数のマーカーの使用を必要とし、可変性は、細胞懸濁液を分析する粒度及び細胞の大きさである。 図1-3に示すマーカーと説明は、培養中の代表的な細胞型のための良い選択肢である。これは、5日目のためにはCD115及びF4 / 80のような他のマーカーを使用することが重要であり、特に後、種々の細胞型を区別することは困難である。 接着性のない条件下での骨髄細胞の拡大を考慮すると、マクロファージの分化を長くすることができる未分化単球を蓄積することができる。マクロファージの分化は、汚染を示している。しかし、デュ唯一の培養物からの非接着細胞を回収しながらでも、超低付着培養表面にその接着性質eは、それらが容易に廃棄することができる。以前の実験1,3では、マクロファージによる汚染は深刻な臨床的副作用をもたらしていない。細胞移植後のマウスの臓器や筋肉の組織学的精密検査は、注入されたマクロファージは、脾臓、肝臓や肺などの臓器に同化することを示した。これらの器官におけるマクロファージの同化は、マウスにおいて観察可能な臨床効果を示さなかった。これらのマクロファージによってトリガー分子機構はさらなる調査のための興味深い質問することができます。 全身に適用された場合、単球などマクロファージは、特に、末梢動脈疾患で動脈形成に影響を与える。そのような塞栓症または大量の全身性炎症の臨床副作用があっても250万人以上の単球の注入後に観察されなかったが、これらの細胞は、炎症および経口をトリガすることができtentially重篤な全身作用を引き起こす。ヒト試験では、これらの効果は、臨床的意義を有する可能性が高い。動脈のトリガは、腫瘍増殖または糖尿病性網膜症、および今後の研究で検討されるべきで注入された細胞溶液のような可能な全身作用に影響を与えることができる。 以前の刊行物は、骨髄からの単球の特性は、末梢血細胞から単離されたものとは異なることができる理由を説明する末梢、血液及び骨髄由来単球の特性の違いを説明する9。 単離方法は、同様に臨床興味深い。採血の代わりにヒト骨髄を抽出し、臨床医学の文脈でより実用的である。それは、動物の福祉の世話をするために、必要に応じて鎮痛を適用することが重要である。静脈内注射のためには、動物と同時に、注射器の両方を扱う実践することが重要である。シングルANIの場合MALは、静脈の品質と尾の皮膚が苦しむ、複数回の注入を受けている。 これらの欠点にもかかわらず、この方法は、単球の挙動および特性を研究するのに非常に有用である。アテローム性動脈硬化症、免疫や炎症の分野での実験は、この方法から利益を得ることができる。わずか30分で、6ウェルの超低取付面プレート上の細胞の播種に骨髄の抽出から必要とされる。このプロトコルの高い収率が細胞ドナーとして必要な動物の数を最小化するため、倫理的な問題は、最小化される。この新しい方法は、単球を含む多くの研究のために参考になります。 我々は、単球の移植を介して、側副血管成長の治療の増強に焦点を当ててきた。動脈形成の増強のための単一の要因のアプローチが入り混じった結果13,14を生成した理由を、このプロセスの多因子性質が説明できるかもしれない。これは、中につながっている細胞ベースの治療のvestigation。自家骨髄由来の幹細胞および前駆細胞は、移植のための潜在的な細胞として同定されているが、中程度の臨床的利益は15報告されている。投与量、単離方法、および投与経路の研究に加えて、さらなる研究が依然として最適な細胞タイプを決定するために必要とされる。自家細胞移植の欠点は、先天的および/または適応免疫応答を活性化することができないこととすることができ、両方が動脈形成のために重要である。局所炎症を増加させると、動脈16のための最高の刺激を表すことができる。 全身性の薬物送達が所望される場合、単球の静脈内注射は、マウスモデル内の細胞移植のための一般的な方法である。なぜなら、全身効果のため、副作用は同様に起こり得る。静脈注射をターゲットにしていることを確認してください。それは、特に重く色素性マイクを注入し、動脈と静脈を、混同することが一般的であるE。動脈注射は、循環における全身の問題につながる塞栓を引き起こす可能性があります。

The isolation of monocytes is important and critical for many in vitro and in vivo studies. These cells are targets for diseases such as peripheral arterial disease, coronary heart disease, or other ischemic diseases, since collateral vessel growth is strongly driven by local inflammation. Inflammatory responses include endothelial activation and local recruitment of leukocytes, mainly monocytes, which then mature to macrophages and create a highly arteriogenic environment by secreting multiple growth factors to induce the remodeling of an arteriole into a functional collateral artery1-3. Monocytes also mature to dendritic cells, which are frequently used for immunological studies4,5 and cancer research6,7.
Problematic in the approach for monocyte isolation from peripheral blood8 is the high number of donor animals needed to produce a sufficient amount of monocytes for most analyses. Former protocols describe methods such as density gradient centrifugation and cell depletion via MACS9 when isolating monocytes; however, these techniques can alter the characteristics and functionality of monocytes which can lead to difficulties in interpretation10,11. Moreover, these methods are difficult and can reduce experimental reproducibility.
Our aim with this protocol is to provide a simple and cost effective method to generate large amounts of bone marrow-derived monocytes. Due to the high cell yield of 11 x 106 &#177; 3 x 106 cells obtained by this protocol, we can substantially reduce the number of mice required during the isolation of bone marrow-derived monocytes. The procedure can be completed within a minimal amount of time, and without using expensive and complicated techniques as referenced above. Here, we extract monocytes from native bone marrow suspension of donor mice, cultivate the suspension on ultralow attachment plates, and supplement the solution with 20 ng/ml M-CFS. On day 5 of incubation, cells are harvested and characterized to confirm functional and phenotypic properties.
For experiments in the field of arteriogenesis, intravenous transplantation of these bone marrow-derived monocytes into mice is an effective method of systemic drug delivery, which can be combined with femoral artery ligation in common peripheral arterial disease models.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>6-well-ultra-low-attachment plate</td>
			<td>Corning Incorporated, NY, USA</td>
			<td></td>
			<td>6-well-ultra-low-attachment plate, with cap, sterile</td>
		</tr>
		<tr>
			<td>8- 12 week old, male, balb/c mice&#160;</td>
			<td>Charles River, Sulzfeld, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well-plate</td>
			<td>Greiner bio one GmbH, Frickenhausen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blue dead cell stain</td>
			<td>Life technologies GmbH, Darmstadt, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumine</td>
			<td>GE Healthcare, Freiburg, Germany</td>
			<td></td>
			<td>Fraction V, pH 7,0</td>
		</tr>
		<tr>
			<td>Canules</td>
			<td>B. Braun, Melsungen AG, Melsungen, Germany</td>
			<td></td>
			<td>28G, 30G</td>
		</tr>
		<tr>
			<td>CD115</td>
			<td>eBioscience, San Diego, USA</td>
			<td>12-1152</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>eBioscience, San Diego, USA</td>
			<td>53-0112</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td></td>
			<td>With cap, steril</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter GmbH, Krefeld, Germany</td>
			<td></td>
			<td>Allegra&#174; X-15R centrifuge</td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet, Mannheim, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disinfection agent</td>
			<td>Sch&#252;lke&#38;Mayr GmbH, Norderstedt, Germany</td>
			<td></td>
			<td>Kodan Tinktur forte</td>
		</tr>
		<tr>
			<td>Disposable scalpel No.10&#160;</td>
			<td>Feather safety razor Co.Ltd, Osaka, Japan&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%&#160;</td>
			<td>Otto Fischar GmbH und Co KG, Saarbr&#252;cken, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Extraction unit Pipetus</td>
			<td>Hirschmann Laborger&#228;te GmbH &#38; Co.KG, Eberstadt, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>AbD Serotec, D&#252;sseldorf, Germany</td>
			<td>MCA497APC</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS buffer&#160;</td>
			<td>Manufactured by our group with single components</td>
			<td></td>
			<td>PBS, 0,5% BSA, 0,1% NaN3</td>
		</tr>
		<tr>
			<td>FACS device</td>
			<td>Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA</td>
			<td></td>
			<td>BD FACS Canto II</td>
		</tr>
		<tr>
			<td>FACS tubes&#160;&#160;&#160;&#160;</td>
			<td>Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon&#174; pipette</td>
			<td>Becton Dickenson Labware, NY, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Rubis, Stabio, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td>R&#246;sner-Matby Meditrade GmbH, Kiefersfelden, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gr1</td>
			<td>eBioscience, San Diego, USA</td>
			<td>53-5931</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating plate&#160;</td>
			<td>Labotect GmbH, G&#246;ttingen, Germany&#160;</td>
			<td></td>
			<td>Hot Plate 062</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Ewald Innovationstechnik GmbH, Bad Nenndorf, Germany</td>
			<td></td>
			<td>Incu safe</td>
		</tr>
		<tr>
			<td>Isofluran</td>
			<td>Baxter Deutschland GmbH, Unterschlei&#223;heim, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Carl Zeiss SMT GmbH, Oberkochen, Germany</td>
			<td></td>
			<td>Axiovert 40 &#176;C</td>
		</tr>
		<tr>
			<td>Macrophage-Colony Stimulating Factor</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/srp3110?lang=de&amp;region=DE">SRP3110&#160;</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Mechanical shaker</td>
			<td>IKA, Staufen, Germany</td>
			<td></td>
			<td>ms2 minishaker</td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>PAA Laboratories GmbH, Pasching, Austria</td>
			<td></td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Micro test tubes</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microbiological work bench</td>
			<td>Thermo Electron, LED GmbH, Langenselbold, Germany</td>
			<td></td>
			<td>Hera safe</td>
		</tr>
		<tr>
			<td>Monocyte wash buffer&#160;</td>
			<td>Manufactured by our group with single components</td>
			<td></td>
			<td>PBS, 0,5% BSA, 2mM EDTA</td>
		</tr>
		<tr>
			<td>Mouse restrainer</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Berlin Chemie AG, Berlin, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaN3 (sodium acide)</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer counting chamber</td>
			<td>Paul Marienfeld GmbH und Co.KG, Lauda-K&#246;nigshofen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon cellsieve</td>
			<td>Becton, Dickinson and Company, Franklyn Lakes, New Jersey, USA</td>
			<td></td>
			<td>Cell strainer, 70 &#181;m mesh size</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma Aldrich, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Life technologies GmbH, Darmstadt, Germany</td>
			<td></td>
			<td>pH 7.4, sterile</td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td></td>
			<td>10&#181;L/100&#181;L/200&#181;L/1000&#181;L</td>
		</tr>
		<tr>
			<td>Pipetting heads</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td></td>
			<td>Cellstar 5 ml, 10 ml</td>
		</tr>
		<tr>
			<td>Suction unit</td>
			<td>Integra bioscience, Fernwald, Germany</td>
			<td></td>
			<td>Vacusafe comfort</td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Word Precision Instruments, Inc., Sarasota, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>B. Braun, Melsungen AG, Melsungen, Germany</td>
			<td></td>
			<td>1mL Omnifix&#174; -F insuline syringe</td>
		</tr>
		<tr>
			<td>Tubes with cap</td>
			<td>Greiner bio one GmbH, Frickenhausen, Germany</td>
			<td></td>
			<td>15mL/50mL Cellstar tubes</td>
		</tr>
		<tr>
			<td>Warm water bath</td>
			<td>Julabo Labortechnik GmbH, Seelbach, Germany</td>
			<td></td>
			<td>Julabo SW22</td>
		</tr>
	</tbody>
</table>

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.

マウス骨髄から抽出された細胞溶液は、様々な細胞型から成る。主要な細胞型は、リンパ球、顆粒球及び単球である。細胞型は、分化の5日後に採取し、ネイティブ懸濁液および細胞については、図1に示されているサイズおよび粒度によって推定することができる。培養中のシフト細胞組成に注意してください。しかし、集団の正確な分類は、細胞マーカーの特徴的な表現に依存しなければならない。 MPS-系の細胞を同定するために使用される最も重要なマーカーは、M-CSF受容体として機能し、特に単球前駆細胞、単球およびマクロファージ上に発現されるCD115である。したがって、独特のMPS-サブセットを識別するために使用されないかもしれないが、確実に、単球/マクロファージ分化に打ち込ま細胞の絶対量を推定することができる。異なる中のCD115発現をタイムラインについては、図2を参照してくださいentiation。 成熟マーカーF4 / 80は、若年性単球、成熟した単球、およびマクロファージを区別するのに便利です。マクロファージは、マーカーに対して陰性である単球前駆細胞を、少年と比較して、F4 / 80の強力な発現を示す。成熟した単球をF4 / 80の中間の発現を示す。したがって、CD115及びF4 / 80マーカーの組み合わせは、（ 図3を参照）、細胞分化を定量化し、監視するための実行可能な方法を表している。 CD115及びF4 / 80の組み合わせは、従来のセルの適用ルーチン培養観察および品質管理のために十分である。培養の5日目に骨髄由来単球のより詳細な検査は、末梢血単核球12のものに一致それらの構造的および機能的特性を確認する。 これは、尾の張力を避け、拘束具に慎重にマウスを修正することが重要です。 freedoを制限注射を容易にするために、呼吸阻害することなく、可能な限りの移動メートル。 <img alt="図1" src="/files/ftp_upload/52347/52347fig1highres.jpg" /> 図1の左パネル：培養中の1日目に細胞型の構成細胞型は、リンパ球、単球および顆粒が挙げられる。右パネル：培養中にシフトする細胞組成に注意してください。細胞集団は、単球およびマクロファージが含まれています。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52347/52347fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/52347/52347fig2highres.jpg" /> 分化中のCD115の発現図2.タイムライン。全ての細胞の&gt; 95％がその広大なことを示す、分化の5日後にCD115陽性であるノート<html>細胞の大部分は、単球またはマクロファージでのいずれかである。分類は、細胞の大きさと細かさに基づいてすることができますが、特定の細胞マーカーは、より信頼性がある。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52347/52347fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/52347/52347fig3highres.jpg" /> / 80球/マクロファージ成熟マーカーF4の発現を増加図3. 5日目：。単球の表現型と一致している中間のF4 / 80発現が、 ​​人口の外観に注意してください。 7日目：マクロファージの成熟と一致して、F4 / 80式の右シフトに注意してください。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52347/52347fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> E 4 &quot;SRC =&quot; /ファイル/ ftp_upload / 52347 / 52347fig4highres.jpg &quot;/&gt; 図4.マウス尾静脈注射のための拘束装置で固定した。後は慎重にマウスを拘束する静脈は背側に表示されるまで、尾を90度回転させる。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52347/52347fig4large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図5" src="/files/ftp_upload/52347/52347fig5highres.jpg" /> 図5.マウスの尾の概略断面。画像はマウスの尾の血管内の場所を示している。動脈（赤）は、腹と背側の側に位置し、静脈は尾の側面にあるされている。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52347/52347fig5large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes at JoVE.com

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

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 ...

isolation-intravenous-injection-murine-bone-marrow-derived

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

23.5K ビュー.  Otto von Guericke University Magdeburg. 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).

ビデオ: Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

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 ...

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 ...

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.

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 ...

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, ...

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 ...

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 ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

コラゲナーゼEを用いてマウスコロンからのラミナプロプリア単核細胞の単核細胞の単核細胞の単核細胞の単核細胞の単核細胞の単核細胞の単核細胞の単核細胞の単核細胞の単核細胞

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...