The research in the Herskovits lab is supported by 335400 ERC and R01A/109048 NIH grants.

注：全実験の間に、マクロファージ細胞を、5％CO 2、強制空気インキュベーター中で37℃でインキュベートされ、唯一のクラスII生物学的安全キャビネット内で実施される実験操作、インキュベーターから取り出し。 L.での作業モノサイトゲネス細菌は生物学的安全性レベル2規則に従っています。 1.細胞の調製および細菌感染（1日目および2） <ol><li> 1日目<ol><li> 30ミリリットルBMDM +ペニシリン-ストレプトマイシンメディア（ 表2）145 mmディッシュ上にシード2.0×10 7骨髄由来マクロファージ細胞（BMDM）。各細菌株のシード3のプレートを分析します。 5％CO 2、強制空気インキュベーター中で37℃で一晩インキュベートします。 </li><li>野生型（WT）L.の一晩の細菌培養を開始モノサイトゲネスは、標準的なインキュベーター中で30℃でのブレインハートインフュージョン（BHI）培地10mlに10403S株。場所培養管を振盪せずに斜め。注意：これらの成長条件は、効率的な感染を促進鞭毛遺伝子の発現をアップレギュレートします。 </li></ol></li><li> 2日目<ol><li> 37℃でのプレ暖かいBMDMの（ペニシリン-ストレプトマイシン抗生物質なし）培地（ 表2）、リン酸緩衝生理食塩水（PBS）。 </li><li>抗生物質を除去するために、予め温めておいたPBS 25mlで二回マクロファージ単層を洗浄します。 30ミリリットルの新鮮なBMDM培地を追加します。 </li><li> 、1分間&gt; 14,000×gで細菌を遠心分離し、上清を廃棄し、ピペッティングによりPBS 1.5 ml中に静かに細菌を再懸濁することにより、PBSで一晩細菌培養の1.5ミリリットルを洗います。二回繰り返します。 </li><li>野生型L.の洗浄一晩培養物0.5mlで各マクロファージプレートに感染モノサイトゲネス 。複数のプレートを使用している場合は、15分間隔で各プレートに感染します。この時間間隔は、感染の終了時に個別に各プレートを収穫できるようになります。 注意：感染の多重度（MOI）を連続希釈をめっきすることによってアッセイされます細菌培養物のSはBHI寒天プレート上での感染のために使用し、37℃でのプレートの24時間のインキュベーション後にコロニー形成単位（CFU）をカウントします。 WT L.の0.5ミリリットルを使用して、 モノサイトゲネスは、〜100のMOIで10403S結果（マクロファージ細胞当たり100細菌CFU）を株。細胞内増殖に欠陥細菌の変異体を分析する場合、より高いMOIを考慮すべきです。 </li><li> 37℃で0.5時間インキュベーションした後、付着していない細菌を除去するために、PBSで2回感染した細胞を洗浄し、30ミリリットル予め温めておいたBMDM媒体を追加します。付着した細菌は、次の0.5時間の間に内部化されます。 </li><li>細胞外細菌を殺すために：（50μg/ mlの最終濃度に達するように千1）1時間後の感染では、ゲンタマイシンの30μlを添加します。 </li><li>真空出口ポートを備えた液体回収フラスコにフィルタヘッドを配置することによって、フィルタ装置を組み立てます。そして、シリンダー漏斗に続くフィルタヘッド、上のフィルター（0.45μm）を配置し、金属Cと異なる部分を確保ランプ。氷のように冷たいRNaseフリー水を準備します。 </li><li>次のように感染した細胞から6時間感染後、収穫菌で。各プレートは個別に扱います。 注：通常、L.モノサイトゲネスは、マクロファージ細胞における感染の6時間の間に成長の1-ログを完了します。短い潜伏期間がかなり低い細菌負荷につながる可能性がありながら、より長いインキュベーションは、細菌異常増殖およびマクロファージの細胞死につながる可能性があります。 MOI感染の時間は、最適条件に到達するために変更することができます。 <ol><li> PBSで1回に感染した細胞を洗浄。マクロファージ細胞を溶解させ、氷冷のRNaseフリー水20ミリリットルを追加します。セルスクレーパーを使用して、迅速しかし慎重にプレートから細胞をこすり取ります。 </li><li> 50ミリリットルコニカルチューブに溶解した細胞を収集します。 30秒間ボルテックス。 4℃で3分間、800×gで遠心分離します。 </li><li>真空システムを使用してフィルタ装置を介して上清を渡します。ピンセットを使用して15mlのCONIに転送迅速フィルタロールとCALチューブ。液体窒素中でフィルタを使用してチューブをスナップ凍結。 </li><li> 1.2.7で説明したように、次のサンプルのための新しいフィルターでろ過装置を組み立てます。 </li><li>次の日のために-80℃で凍結したフィルタを格納します。あるいは、核酸抽出に直接進みます。 </li></ol></li></ol></li></ol> 2.核酸抽出（3日目） 注：ドラフト内でフェノール及びクロロホルムソリューションですべての操作を実行します。 <ol><li>酸性フェノールの1ミックス：クロロホルム、各サンプルの400μlの1を準備します。別々のチューブに混合し、その後吸引により得られた水層を撤回。 10％SDSを40μlのを追加します。 </li><li>冷たいそれらを保つために氷の上にフィルタ収容管を解凍します。各フィルタを含むチューブに酢酸EDTA（AE）のバッファ（ 表2）の650μlを添加します。 </li><li>可能な限り迅速に次の手順を実行します。 <ol><li>積極的にそのようにフィルタを含むチューブをボルテックスフィルタは、管の周囲に案内いたしますと、バッファは完全にフィルターを洗うこと。常に氷上に戻ってそれを置くことによって、チューブの寒さを保ちます。注：反転しながら、さらに、完全にオフフィルタ細菌を洗浄するために、チューブをボルテックスする必要があるかもしれません。 </li><li>すべてのチューブのために繰り返します。スピンダウンするまもなく懸濁液（120×gで1分間）プロセスの終了時に有用であり得ます。 </li></ol></li><li> SDSおよびフェノール/クロロホルムミックス（2.1で調製した）含む1.5 mlマイクロチューブに細菌含有緩衝液を移します。すべてのチューブのために繰り返します。フィルターから残った液を得るために再びチューブを含むフィルター（120×gで1分間）をスピンする必要があるかもしれません。 </li><li>マルチチューブボルテックスデバイス内のすべての1.5ミリリットルチューブを置き、10分間フルスピードでボルテックス。 </li><li> 10分間65℃のヒートブロック中で管をインキュベートします。 5分間最大速度で遠心分離（&gt; 14,000 XG）。 </li><li>各トンから水層（約400μl）を転送3 M酢酸ナトリウム（pH 5.2）1.0 mlで100％エタノールの40μLを含む新しい1.5mlチューブにUBE。徹底的に各チューブをボルテックス。 注：グリコーゲンを沈殿ペレットの可視化を向上させるために、この段階で添加することができます。 </li><li> 1時間-80℃でサンプルをインキュベートします。また、Cで一晩-20℃でサンプルをインキュベートします。そして、最大速度（&gt; 14,000 XG）で20分間4℃でサンプルを遠心します。 </li><li>慎重に各チューブからのエタノール（上層）を吸引します。徹底的に各サンプルと渦に500μlの冷70％エタノールを追加します。最高速度（&gt; 1 4000 XG）で20分間4℃で遠心分離します。 </li><li>慎重に各チューブからエタノールを吸引します。加熱なしで真空蒸着装置を用いて約2分間のサンプルを乾燥させます。サンプルを過乾燥させないでください。 </li><li>各サンプルに25μlのRNaseフリー水を追加します。 20分間室温でインキュベートします。慎重にボルテックスとスピンダウン。 </li><li> T中のRNA濃度を測定微量紫外可視分光光度計を使用して彼のサンプル。 1μgのプレート当たりの核酸 - 約0.5を抽出することを期待しています。 注：技術的反復は、この段階で組み合わせることができます。 </li></ol> 3. DNアーゼ治療<ol><li>マイクロチューブ内の表1に従って反応を設定します。 45分間37℃でインキュベートします。最大速度で2分間遠心分離することにより、450μlのRNaseフリー水とフェノール-クロロホルム- IAAミックス（ 表2）500μlの、別々の相を追加します（&gt; 14,000 XG）。 </li><li>新しいチューブに水層を移し、500μlのクロロホルム- IAAミックス（ 表2）を追加し 、最大速度で2分間の遠心分離によって渦と別々の相（&gt; 14,000 XG）。 </li><li>新しいチューブに水層を移し、エタノール1mlの3 M酢酸ナトリウム（pH5.2）の50μLを加えます。渦。 注：グリコーゲンを沈殿ペレットの可視化を向上させるために、この段階で添加することができます。 </li><li&gt;は-80℃で1時間インキュベートします。また、Cで一晩-20℃でサンプルをインキュベートします。最大速度で20分間4℃で遠心分離します。 </li><li>慎重に各チューブからエタノールを吸引します。徹底的に各サンプルと渦に500μlの冷70％エタノールを追加します。最高速度で20分間4℃で遠心分離します。慎重に各チューブからエタノールを吸引します。 </li><li>加熱なしで真空蒸着装置を用いて約2分間のサンプルを乾燥させます。サンプルを過乾燥させないでください。 </li><li>室温、ボルテックス、およびスピンダウンで2分間インキュベートし、RNaseフリー水12μlのを追加します。サンプルは、精製されたRNAが含まれている氷の上に保管してください。あるいは、0.1mMのEDTAまたはTE緩衝液（10mMのTris、1mMのEDTA）を用いてRNaseを含まないH 2 O中のRNAを溶かします。 </li><li>微量紫外可視分光光度計を用いてRNA濃度を測定します。 （技術的反復を併用した場合）、サンプルあたりのRNAの〜100 ngのを期待しています。 注：このプロトコルに従って抽出したRNAは、foのに適しています多数の技術によりR遺伝子の転写解析。我々は通常、Lを研究するためにRT-qPCR分析を採用しますマクロファージ細胞1-4の感染の間に遺伝子転写をモノサイトゲネス 。 </li></ol>

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The authors have nothing to disclose.

ここで説明するプロトコルは、Lからの細菌RNAの単離のために最適化された方法を表しマクロファージ細胞において細胞内で増殖する細菌をモノサイトゲネス 。このプロトコルは、細胞微分溶解に基づいており、細菌RNAの濃縮のための2つの主要なステップ含んでいる：遠心分離を用いてマクロファージ核沈殿、濾過による細菌の迅速な収集を。これらのステップは、標準のRNA抽?...

細胞内細菌性病原体─感染症とヒト宿主の内部細胞を増殖し、再生することができるの原因となる細菌は─世界の主要な健康問題5です。侵入し、哺乳動物細胞内で複製するには、細胞内病原体は、洗練された病原性のメカニズムと要因を取得しています。これらのメカニズムは、疾患を引き起こす能力の基本であるが、我々は彼らの規制とダイナミクスについて少し知っています。液体培地中で増殖させた細菌の遺伝子発現プロファイルは、宿主細胞内の実際の環境を反映していないので、それらの細胞内ニッチで増殖した細菌のトランスクリプトーム解析の必要性が高まっています。このような分析は、ホストによってトリガーした特定の細菌の適応の解読を可能にし、治療設計のための新しいターゲットを識別するのに役立ちます。哺乳動物のRNAがでによって細菌RNAを上回っているため、細胞内で増殖させた細菌のトランスクリプトーム解析は非常に困難です少なくとも10倍。本稿では、我々は、マウスマクロファージ細胞内で成長しているリステリア菌の細菌から細菌RNAを単離するための実験方法を説明します。抽出されたRNAは、RT-PCR、RNA-のSeq、マイクロアレイおよび他のハイブリダイゼーションに基づく技術のような転写分析の様々な技術によって、病原性細菌の細胞内の適応と毒性メカニズムを研究するために使用することができます。 リステリア菌は、ヒトのリステリア症の原因物質で、主に免疫無防備状態の個体、高齢者や妊娠中の女性6をターゲットに臨床症状を伴う疾患です。それ 7を研究する宿主-病原体相互作用でモデルとして何十年も使用されてきた哺乳動物細胞の広い配列に侵入グラム陽性の通性細胞内病原体です。侵略の際に、それはトンにエスケープする必要があり、そこから液胞または（食細胞の場合）ファゴソームに最初に存在します彼は、複製するために細胞質ゾルをホストします。いくつかの病原性因子は、エスケープ処理、主に細孔形成溶血素、リステリオリシンO（LLO）および2つの追加のホスホリパーゼ8を媒介することが示されています。細胞質ゾル中の細菌は細胞内のアクチンフィラメント上で自分自身を推進すると（ 図1）細胞から細胞へ拡散するためにホストアクチン重合の機械を使用しています。 L.のすべての主要な病原性因子侵入に関与モノサイトゲネス 、細胞内生存および複製、マスター病原性転写調節因子、PRFAによって活性化される。8-10。 この10年間、私たちと他の人が行ったいくつかの研究は、正常宿主細胞2,11-15内部の細胞内で増殖させた細菌のトランスクリプトーム解析のための方法を適用しています。 diffeによる細菌RNAの1）選択的濃縮および2）RNA単離：二つの主要なアプローチが基づいているホスト-RNAから細菌RNAを分離するために使用されますrential細胞溶解。最初のアプローチは、（市販のキットを用いて、例えば）は、哺乳動物RNA分子または細菌転写配列（SCOTS）11を選択的に捕獲する全RNA抽出物のサブトラクティブハイブリダイゼーションに依存しています。第二のアプローチは、細菌細胞が無傷のままで宿主細胞が溶解された細菌宿主細胞の示差溶解に依存します。バクテリア細胞は、次いで、通常、遠心分離によって、宿主細胞溶解物から分離され、そしてRNAは、標準的な技術を使用して抽出されます。このアプローチを使用して主な問題は、一緒に完全な細菌を、宿主細胞の核は、したがって、RNA調製物はまだ、哺乳類RNAを含んでも単離されていることです。この問題を克服する一つの方法は、この手順は、通常、抽出中の遺伝子発現プロファイルの変化の懸念を高める時間を要するものの、分画遠心法を用いて、宿主細胞の核から無傷の細菌を分離することです。本稿では、改善されたと迅速なBAを提示します細胞差動溶解アプローチに基づいているcteria RNA抽出プロトコル。まず、L.モノサイトゲネス感染マクロファージ細胞を冷水で溶解します。次に、マクロファージ核を短時間の遠心分離によって除去され、無傷の細菌が急速にそのRNAからの細菌の核酸のホットフェノールSDS抽出を使用して単離し、フィルター上に収集されます。

<table>
	<tbody>
		<tr>
			<td rowspan="2">Listeria monocytogenes 10403S</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">20</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Bone marrow derived macrophages prepared from C57B/6 female mice</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">21</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>H2O, RNAse free</td>
			<td>Thermo Scientific</td>
			<td>10977-015</td>
			<td>DEPC-treated water can be used</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>41965039</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Gibco</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360-088</td>
			<td></td>
		</tr>
		<tr>
			<td>b-Mercaptoethanol&#160;&#160;</td>
			<td>Gibco</td>
			<td>31350010</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma-Aldrich</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline-PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain heart infusion (BHI)</td>
			<td>Merckmillipore</td>
			<td>1104930500</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol saturated pH 4.3</td>
			<td>Fisher</td>
			<td>BP1751I-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher</td>
			<td>BP1145-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Iso-amyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>W205702</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>W302406</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>EDS</td>
			<td></td>
		</tr>
		<tr>
			<td>DNaseI</td>
			<td>Fermentas,</td>
			<td>EN0521</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS 10%</td>
			<td>Sigma-Aldrich</td>
			<td>L4522</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Merck Millipore</td>
			<td>1070174000</td>
			<td></td>
		</tr>
		<tr>
			<td>37&#176;C, 5% CO2 forced-air incubator</td>
			<td>Thermo Scientific</td>
			<td>Model 3111</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scrapers</td>
			<td>Nunc</td>
			<td>179693</td>
			<td></td>
		</tr>
		<tr>
			<td>Kontes glass holder for 45 mm filters</td>
			<td>Fisher</td>
			<td>K953755-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>MF-Millipore filters 45 mm, 0.45 &#181;m</td>
			<td>Merck Millipore</td>
			<td>HAWP04700</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac system</td>
			<td>Thermo Scientific</td>
			<td>SPD131DDA</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex-Genie 2</td>
			<td>Scientific Industries</td>
			<td>Model G560E</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>145 mm cell culture dishes</td>
			<td>Greiner</td>
			<td>639 160</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 ml tubes, RNase-free</td>
			<td>Axygen</td>
			<td>MCT-175-C</td>
			<td></td>
		</tr>
		<tr>
			<td>30&#176;C incubator</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>65 &#176;C heat block</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 &#176;C table centrifuge</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile pipettes, 25 ml</td>
			<td>Greiner</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes, 50 ml</td>
			<td>Greiner</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

rna purification from intracellularly grown listeria monocytogenes in macrophage cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

モデルシステムは、 図1に示され、L.に感染したマクロファージ細胞を含みますマクロファージ細胞質ゾル中で複製モノサイトゲネス菌 、。 図2は実験スキームを表す。 図3は、WT L.中の病原性遺伝子のようなRT-qPCR分析の典型的な結果を表します豊富な実験室培地BHIの成長と比較して、マクロファージの成長を<...

Watch this Scientific Journal Video about RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells at JoVE.com

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA精製は、細胞内から成長します<em&gt;リステリア菌</em&gt;マクロファージ細胞で

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

rna-purification-from-intracellularly-grown-listeria-monocytogenes

Research

JoVE Journal

Immunology and Infection

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

9.1K ビュー.  Tel Aviv University. Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

ビデオ: RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

共培養モデルの緑膿菌</emライブヒト気道細胞上に成長した>バイオフィルム

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

免疫・感染学

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

に感染したマウスにおける細菌負荷と免疫応答の測定リステリア菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

感染細胞のクロマチンからのインフルエンザウイルスのリボ核タンパク質複合体のアフィニティ精製

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

病原体胞の精製から<em&gt;レジオネラ菌</em&gt;感染した食細胞

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

の口頭伝達<em&gt;リステリア菌</em&gt;汚染された食品の摂取を介したマウスにおける

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

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 ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

細菌性病原体による細胞の感染を調査するためにイメージングInlC分泌<em&gt;リステリア菌</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

単離、同定、およびマウス胸腺上皮細胞の精製

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

リステリア単球遺伝子を用いた感染マウスにおける培養および心臓コロニー形成における心臓細胞の細菌浸潤の評価

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

と実験感染<em&gt;リステリア菌</em&gt;ホストインターフェロンγ応答を研究するためのモデルとして、

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

RNA精製は、細胞内から成長します

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共培養モデルの緑膿菌バイオフィルム

に感染したマウスにおける細菌負荷と免疫応答の測定リステリア菌

感染細胞のクロマチンからのインフルエンザウイルスのリボ核タンパク質複合体のアフィニティ精製

病原体胞の精製から

の口頭伝達

骨髄由来マクロファージの生産

細菌性病原体による細胞の感染を調査するためにイメージングInlC分泌

単離、同定、およびマウス胸腺上皮細胞の精製

リステリア単球遺伝子を用いた感染マウスにおける培養および心臓コロニー形成における心臓細胞の細菌浸潤の評価

と実験感染