Aging-Associated 病、ケルン大学、ドイツの細胞ストレス反応にケルンの卓越性クラスターからの資金調達でロビンソンのラボで研究をサポート (CECAD; エクセレンス イニシアチブは、ドイツ連邦内 DFG によって資金を供給し、国家の政府) とドイツ研究振興協会 (SFB 670)、ケルン富と、ドイツのケルン大学のマリア ペッシュ財団からの助成金。

病原性 s. の使用を含むすべての手順ネズミチフス菌 は、BSL 2 またはより高い生物学的セキュリティ レベルの施設で実施する必要があります。文化と S のコーティングネズミチフス菌として骨髄由来マクロファージ (BMDMs) の感染は、汚染を防ぐための層流フードの下で行う必要があります。S の分離ネズミチフス菌-ファゴゾームを含む任意 BSL 2 実験室ベンチで実行できます。
 骨髄のマクロファージへの分化のマウスからの抽出され動物の福祉制度のガイドラインに沿って行われ、自然、ノルトライン = ヴェストファーレン州の機関によって承認されました。環境および消費者保護 [Landesamt für Natur、環ならびにノードラインヴェストファーレン州 Verbraucherschutz (LANUV);ファイル: 84 02.05.40.14.082、84 02.04.2015.A443] とケルン大学。
 1 養殖 s <ol><li>Inoculate s.。ネズミチフス菌(SL1344 株) 細菌のループを使ってブレインハートインフュー ジョン (BHI) 5 mL に単一の細菌のコロニーから</li>。
	<li>揺れで一晩 37 ° c の定温器の細菌懸濁液をインキュベートします</li>。
	<li>次の日に 19 mL 三角フラスコに BHI ブイヨンの細菌懸濁液の 1 mL に転送し、, 振動インキュベーターで 37 ° C で</li>。
	<li>監視 s.600 の光学濃度 (OD) を測定することによって成長を ネズミチフス菌 nm (外径 600) 分光光度計。測定すべき約 30 分ごとに</li>
	<li>とき外径 600 1.0 に達する、インキュベーターと 50 mL のチューブに転送から細菌懸濁液を取り外します。4 細菌 15 分 5,400 × g で遠心分離機 ° C</li>
	<li>上清を除去し、滅菌リン酸緩衝生理食塩水 (PBS) の 10 mL で再懸濁します</li>。
	<li>4 ° C で 15 分間 5,400 × g で遠心します。</li>
	<li>上澄みを除去し、4.9 mL の滅菌 PBS 中の細菌を再懸濁します</li>。
</ol> 2。NHS ビオチン/ストレプトアビジン化磁気ビーズの サルモネラ のコーティング ソリューション (NHS ビオチンはジメチルスルホキシド、DMSO に溶解) 細菌懸濁液に作りたてのリンク <ol><li>10 mg/mL の追加の 100 μ L ビオチン前の手順で準備します。たとえば、滅菌 DMSO 0.5 mL に NHS ビオチン 5 mg を希釈します。上下に数回ピペッティングにより正しくミックス</li>。
	<li>5 1.5 mL チューブにミックスを分割します</li>。
	<li>350 rpm で一定の揺れで、サーモに室温 (RT) で 2 時間加温します</li>。
	<li>RT で 10 分間 15,000 × g でチューブを遠心し、上澄みを廃棄します</li>。
	<li>1 mL の滅菌 PBS、15,000 × g で RT で 10 分間遠心で再懸濁します、上澄みを廃棄します</li>。
	<li>余分なビオチン ソリューションのリンクを完全に削除する手順 2.5 を 2 回繰り返します</li>。
	<li>最後の洗浄後に、1 mL の滅菌 PBS でペレットを再懸濁します</li>。
	<li>1.5 mL チューブに 10 mg/mL 磁気ビーズのストレプトアビジン共役溶液 100 μ L を転送し 5 分間磁気ラックにそれを残す</li>
	<li>ピペットで溶媒を除去、磁気ラックからストレプトアビジン - 共役磁気ビーズ ソリューションを取るし、2.7 準備したビオチン コーティング細菌懸濁液の 1 mL でそれを再懸濁します</li>。
	<li>350 rpm で一定の揺れで、サーモに RT で 1 時間加温します</li>。
	<li>磁気ラック ソリューションを配置; 5 分待つし、ピペットと壁に付着した細菌を削除 (レッテルをチューブでそれを保つ &#34; 非コーティング細菌 &#34;)。マグネットに接触して管の壁に付着した分数はビオチン/ストレプトアビジン コーティング細菌</li>。
	<li>磁気ラックからラベル付きの細菌を含むチューブを削除し、1 mL の滅菌 PBS に再懸濁します</li>。
	<li>磁気のラックに再びそれを置き、5 分待つし、ピペットを使用して PBS を削除します</li>。
	<li>ストレプトアビジン共役磁気ビーズでコーティングされていないすべての細菌を除去する手順 2.13 と 2.14 2 回以上を繰り返します</li>。
	<li>最後の洗浄後、コーティング細菌を 500 μ L の滅菌 PBS に再懸濁し、ラベルとして管 &#34; 菌をコーティング &#34;.</li>
	<li>カウント コロニー形成単位 (cfu) 両方の &#34; 非コーティング細菌 &#34; と &#34; 菌をコーティング &#34; BHI 寒天培地プレート上のシリアル希薄をめっきによってソリューション。約 2 × 10 8 cfu/mL のコーティングされた細菌は、外径 600 1.0 の初期の細菌懸濁液 20 mL から取得されます。マクロファージに感染する次の日に使用する 4 ° C でコーティングした細菌懸濁液を維持します</li>。
</ol> 3。BDMDs の感染 <ol><li>ビオチンとストレプトアビジン共役磁気ビーズの細菌のコーティングと同じ日にプレート完全に差別化された BMDMs 22 皿あたり 5 x 10 の 6 セルの密度で 6 cm 料理</li>。
	<li>次の日 4時 5 分 15,000 × g でコーティングされた細菌懸濁液を遠心分離機 ° C</li>
	<li>上澄みを除去し、10 x 10 6 cfu/mL の濃度 10% 牛胎児血清 (FBS) ロズウェル パーク記念研究所 (RPMI) 培で再懸濁します</li>。
	10、感染 (MOI) の多様性でマクロファージに感染する <li>は、BMDMs からメディアを取り出して、準備コーティング細菌懸濁液 5 mL を加えます。すべてのセル型や分離のファゴゾームの実験目的のため最適な MOI の評価がある</li>。
	<li>貪食を同期する 10 分間常温加温します</li>。
	<li>貪食を開始する 30 分間 5% CO 2 インキュベーターで 37 ° C に加温します。他のセルタイプが細菌の内面化を確保するため異なるインキュベーション時間を必要があります</li>。
	<li>料理から細菌を含む培地を外し、非内面の細菌を除去する RPMI でセルを 3 回洗浄します</li>。
	<li>任意の非貧する細菌を殺すために 10 %fbs および 50 μ G/ml のゲンタマイシンを含む RPMI の 10 mL を最後に追加します</li>。
	<li>では、目的の時点までの 5% CO 2 と 37 ° C で感染した BMDMs 孵化させなさい。目的の時点は、分析の目的と使用する携帯型細菌によると実験的に決定する必要があります。S の初期エンドソーム-ファゴソーム融合事象の研究ネズミチフス菌感染 BMDMs 30 分インキュベーションをお勧めします。後の事象の分析、2 時間または 4 時間後ファゴゾームを抽出できます。S と大食細胞の長期培養24 時間を超えてネズミチフス菌はマクロファージの死の結果します。ファゴソーム抽出可能性がありますおそらくビオチン分子の劣化につながる前に細胞内感染症を拡張します。ただし、24 時間まで見られなかったコーティング細菌でビオチンの損失</li>
</ol> 4。S の分離ネズミチフス菌-ファゴゾームを含む <ol><li>準備必要ファゴソームのボリューム分離バッファー (50 mM 管、50 ミリメートル MgCl 2, 5 mM グリコールエーテルジアミン四酢酸 pH.7) ジチオトレイトール (DTT) を最終濃度 1 mM、サイトカラシン B 決勝に追加することによって10 μ M、プロテアーゼとホスファターゼ阻害剤の製造元の推奨どおり濃度 。
	注: DTT、サイトカラシン B を加えなければなりませんファゴソーム分離バッファー A に常に直前に使用します。サイトカラシン B 破壊細胞骨格、細胞膜の破壊を促進する</li>。
	<li>希望の時間にポイントし、感染細胞からメディアを取り出して、右に温めた滅菌 PBS で洗浄</li>
	<li>Phagosom の追加 750 μ Le 分離は一皿 A バッファーに格納し、氷の上の 20 分を孵化させなさい。分離バッファー A のボリュームを比例して調整する必要があります別のセル番号を使用するときです</li>。
	<li>(50 mM のパイプ、pH.7、50 mM MgCl 2、グリコールエーテルジアミン四酢酸 5 mM、220 mM、マンニトールと 68 mM ショ糖) ファゴソーム分離バッファー B の追加 250 μ L。ロック プレート皿の完全な表面をバッファーに届くように。分離バッファー B のボリュームを比例して調整必要があります別のセル番号を使用している場合</li>。
	<li>そっとゴム警官を使用して削ってお皿からセルを削除し、中古冷蔵 1.5 mL チューブに転送します</li>。
	<li>は、26 G 針、少なくとも 15 回 1 mL 注射器を使用して (1 つの吸引プラス 1 回の細胞懸濁液の数の 1 つ放出) を細胞懸濁液を通過します。これは BMDMs のゾル性細胞質のコンテンツをリリースするのに十分です。針通過の数は、細胞の種類ごとに最適化する必要が</li>。
	<li>磁気ラックに細胞懸濁液、5 分を待ちます。磁石に粒子がコーティング-s. を含むファゴゾームです。ネズミチフス菌。懸濁液には細胞成分の残りの部分が含まれています</li>。
	<li>というレッテルを 1.5 mL チューブに懸濁液を転送 &#34; 細胞質 &#34;.</li>
	<li>磁気ラックから隔離されたファゴゾームとチューブを削除し、それらを 1 mL の滅菌 PBS に再懸濁します</li>。
	<li>磁気ラックにファゴソーム サスペンションを置き、5 分待つし、PBS を削除します</li>。
	<li>4.9 と 4.10 分離 米 を洗う手順を繰り返ネズミチフス菌-ファゴゾームを含むします</li>。
	<li>は最後に PBS を削除し、必要なバッファー; たとえば、タンパク質解析のための radioimmunoprecipitation 検定法 (RIPA) バッファー、ファゴゾームを再懸濁します。細胞の初期量と感染の慣性モーメントによって、このプロトコルを次のサンプルあたり約 50-200 μ g のタンパク質を取得します</li>。
</ol>

<ol>
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G., Deyoe, B. L., Cheville, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+and+intracellular+localization+of+Brucella+spp.+in+Vero+cells:+fluorescence+and+electron+microscopy.">Entry and intracellular localization of Brucella spp. in Vero cells: fluorescence and electron microscopy.</a> Vet Pathol. 27 (5), 317-328 (1990).</li><li>Arenas, G. N., Staskevich, A. S., Aballay, A., Mayorga, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+trafficking+of+Brucella+abortus+in+J774+macrophages.">Intracellular trafficking of Brucella abortus in J774 macrophages.</a> Infect Immun. 68 (7), 4255-4263 (2000).</li><li>West, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR+signalling+augments+macrophage+bactericidal+activity+through+mitochondrial+ROS.">TLR signalling augments macrophage bactericidal activity through mitochondrial ROS.</a> Nature. 472 (7344), 476-480 (2011).</li><li>Li, Q., Jagannath, C., Rao, P. K., Singh, C. R., Lostumbo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+phagosomal+proteomes:+from+latex-bead+to+bacterial+phagosomes.">Analysis of phagosomal proteomes: from latex-bead to bacterial phagosomes.</a> Proteomics. 10 (22), 4098-4116 (2010).</li><li>Rao, P. K., Singh, C. R., Jagannath, C., Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systems+biology+approach+to+study+the+phagosomal+proteome+modulated+by+mycobacterial+infections.">A systems biology approach to study the phagosomal proteome modulated by mycobacterial infections.</a> Int J Clin Exp Med. 2 (3), 233-247 (2009).</li><li>Rogers, L. D., Foster, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+phagosomal+proteome+and+the+contribution+of+the+endoplasmic+reticulum.">The dynamic phagosomal proteome and the contribution of the endoplasmic reticulum.</a> P Natl Acad Sci USA. 104 (47), 18520-18525 (2007).</li></ol>

著者が明らかに何もありません。

新しいSの分離法ネズミチフス菌-ここで説明はファゴゾームを含むビオチンとストレプトアビジン共役磁気ビーズ細菌をコーティングします。細胞膜の穏やかな中断の後、磁気ラックを使用して細菌を含むファゴゾームできます簡単に抽出しました。示す細菌の炎症を誘発する病原体の能力を維持宿主細胞の貪食のプロパティは変更されません。このメソッドによって得られる?...

マクロファージ循環専門の貪食細胞検出、巻き込む、および任意の異物侵入の細菌など微生物細胞のアポトーシスに至る末梢の組織内に存在の低下しています。一般微生物 (病原体関連分子パターンまたは PAMPs として知られている) の表面に存在する病原体特定マーカーの表面受容体を介した認識時にマクロファージを開始の細胞膜における複雑な再編囲むと病原体1を phagocytize の順。
包まれて病原体、ファゴソームと呼ばれる細胞内小胞のマクロファージによって含まれています。エンドソーム、リソソームなど他の小胞の分裂と融合の一連、病原体を含むファゴソームは組の phagosomal コンテンツの除去に必要な蛋白質を取得します。したがって、ファゴソームの酵素の組成は、ファゴソーム成熟2として知られているこのプロセスの過程で非常に可変です。
直後に貪食、エンドソーム3融解によるファゴソーム膜を組み込む複雑な空胞 ATPase (V-atpase) ポリコームタンパク。この複合体は、ファゴソーム4ルーメンに細胞質からポンプのプロトンに ATP を利用しています。ファゴソームの酸性化は、5その他の小胞の融合イベントおよび pH 依存分解酵素6の偉大な数の活性化が不可欠です。別ポリコームタンパク酵素複合体ファゴソーム膜上に組み立てはすぐに NADPH オキシダーゼ (NOX) 複合体であります。複雑な NOX は、ファゴソームの内腔に分泌して包まれて微生物7の殺害に大幅に貢献する活性酸素種 (ROS) を生成するために NADPH を酸化させます。
成熟の最初の手順では、ファゴゾームは通常 Rab5 など v ATPase8V0サブユニットと共にそれぞれの初期と後期エンドソームの Rab7 マーカーを提示します。リソソームと後期エンドソーム ファゴゾームの融合貧するの病原体のさまざまな加水分解酵素カテプシン プロテアーゼ、リパーゼ、β-ガラクトシダーゼ9などへの暴露で起因します。内腔の酸性化もこれらの酵素の活性化のために必要です。たとえば、アクティブな短いフォームを生成するカテプシン D の胸の谷間は pH 依存性10です。これらの酵素は、病原体が低下してマクロファージ主要組織適合遺伝子複雑な (MHC) クラス II 分子11適応免疫応答をトリガーする T 細胞に提示された病原体由来のペプチドの生産を仲介します。
したがって、ファゴソーム成熟は生得の免疫反応のため非常に重要ですし、自然免疫と適応免疫システムの腕をリンクします。病原体はファゴソーム成熟の上記プロセスを介してマクロファージによって除去を克服する戦略を進化している驚きではないです。たとえば、細胞内細菌結核菌、レジオネラ ・ ニューモフィラ防止抑制 V-atpase アセンブリとそれに伴う内腔の酸性化12,13 によるファゴソーム成熟.リステリア菌や赤痢菌など他の細菌は、細胞質14,15に脱出するファゴソーム膜の細孔形成を誘発します。その一方で、サルモネラ血清型ネズミチフス菌(S。ネズミチフス菌)そのレプリケーション16の適切な場所に変換する液胞内ファゴソームのプロパティを変更することです。この機能によって、 s.ネズミチフス菌ファゴソーム成熟の病原体を介した干渉を研究する非常に興味深いモデル。
ネズミチフス菌は通性細胞内細菌人間の胃腸炎を引き起こすです。粘膜、 s.の侵略の後ネズミチフス菌迅速検出し貧するの大食細胞によっておよびファゴゾーム17の内で含まれています。いくつかのレポートはそのs.を前述しました。ネズミチフス菌-エンドソーム、リソソームは18メーカーで示し、他の研究は、米に防いだファゴソームとリソソームの融合を発見した含んでいる phagosomeネズミチフス菌感染症19。
S.によって最初に、ファゴソーム成熟蛍光顕微鏡による感染症について調べた。細菌を含む分離技術の開発ファゴゾームはエンドソームやライソソーム マーカー ファゴソーム内容のより正確な研究を有効にします。までに、主要な細菌を含む分離使用方法ファゴゾームはショ糖ステップ グラデーション18,20細胞レベル下の分別。ただし、このメソッドは、ファゴゾームに機械的な損傷を引き起こす可能性が、phagosomal コンポーネント (蛋白質・脂質) の安定性に影響を与えることができます、時間がかかる複数の遠心分離手順を必要です。また、それは、超遠心機の使用を必要です: は、アクセスできないすべての研究室の専門機器の一部。
最近では、新しいアプローチは、細菌を含む分離に適用されている phagosome、ビオチン化 lipopetide (Lipobiotin) と後に細菌性病原体は、ラベル付けは、ストレプトアビジン共役磁気ビーズ21 を使用して抽出.磁気ビーズのストレプトアビジン共役続いて NHS ビオチンと細菌表面アミン含有高分子の分類によって相補的な方法を提案します。本法で得られたファゴゾーム エンドソームやライソソーム マーカーで濃縮され非常とアッセイ、オミックス解析タンパク質解析からの広い範囲に使用できます。さらに、ultracentrifuges などの特殊な機器は不要です。また、遠心分離手順を排除し、ファゴゾームに機械的な損傷や採用時の量が大幅に削減します。このメソッドは、グラム陽性球菌、この原稿にも含まれているなど、他の細菌を含むファゴゾームの隔離のために簡単に対応できます。要約すると、このSの分離法ネズミチフス菌-ファゴゾームを含む、シンプルでコスト効率の高い、ショ糖で古典的な分離より時間もかかります以下勾配遠心、高度のレンダリング濃縮細菌含むファゴゾーム。

<table>
	<tbody>
		<tr>
			<td>EZ-Link NHS Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>20217</td>
			<td></td>
		</tr>
		<tr>
			<td>FluidMag Streptavidin</td>
			<td>Chemicell</td>
			<td>4205</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Carl Roth</td>
			<td>9156.2</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Carl Roth</td>
			<td>A537.4</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Carl Roth</td>
			<td>3054.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Carl Roth</td>
			<td>4621.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Carl Roth</td>
			<td>4175.1</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma</td>
			<td>43816</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease and Phosphatase inhibitor cocktail</td>
			<td>Thermo Fisher Scientific</td>
			<td>1861280</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochalasin B</td>
			<td>Sigma</td>
			<td>C6762</td>
			<td></td>
		</tr>
		<tr>
			<td>DYNAL or DynaMag Magnet</td>
			<td>Thermo Fisher Scientific</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>SmartSpec 3000 Spectrophotometer</td>
			<td>Bio-Rad</td>
			<td>170-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial loop (10&#181;l)</td>
			<td>Sarstedt</td>
			<td>86.1562.010</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmonella enterica serovar Typhimurium SL1344</td>
			<td>Leibniz Institute DSMZ-German collection of Microorganisms and Cell Cultures</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Biochrom</td>
			<td>FG1415</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Biochrom</td>
			<td>L1825</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy5-streptavidin</td>
			<td>Invitrogen</td>
			<td>SA1011</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-beta-actin antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-47778</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mCherry antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>PA5-34974</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Rab5 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-46692</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Rab7 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-10764</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-v-ATPase (V0) antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-28801</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-v-ATPase (V1) antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-20943</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-cathepsin D antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-6486</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Tomm20 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-17764</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-calnexin antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-46669</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GAPDH antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-20357</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of salmonella typhimurium-containing phagosomes from macrophages

サルモネラは、人間の胃腸炎を引き起こす通性細胞内細菌です。粘膜、S.の侵略の後ネズミチフス菌迅速検出し貧するの大食細胞によって、低下するためファゴゾームと呼ばれる小胞に含まれています。Sの分離ネズミチフス菌-含むファゴゾームは、広く研究に使用されているどのようにs.感染症は、細菌の劣化を防ぐためファゴソーム成熟のプロセスを変更します。細菌を含む分離従来、ファゴゾーム ショ糖勾配遠心法によって実行されました。ただし、このプロセスは時間がかかり、専用の装置とある程度の器用さが必要です。Sの分離の簡単かつ迅速な方法は、ここで説明しました。ネズミチフス菌-ビオチン-ストレプトアビジン共役磁気ビーズの細菌をコーティングすることによりマクロファージからファゴゾームを含みます。分離ファゴゾーム アッセイ、脂質、代謝、タンパク質解析などの広い範囲のための利用可能の選択の任意のバッファーで本法で得られたファゴゾームを中断できます。要約すると、このSの分離法ネズミチフス菌の特定、効率的、迅速なファゴゾームを含む最低限の機器を必要とする、古典的なショ糖勾配超遠心分離法よりも汎用性が。

細菌を含む分離このプロトコルによってファゴゾーム細菌の最初のステップとしてビオチン化が必要です。我々 したがって、 s.の有効性を評価ビオチン標識に感染した BMDMs の共焦点顕微鏡解析によるネズミチフス菌ビオチン化 mCherry-S。ネズミチフス菌Cy5 ストレプトアビジンが付いた。簡単に、BMDMs は、mCherry-Sとこのプロトコルで説...

Watch this Scientific Journal Video about Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages at JoVE.com

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

簡単で迅速なサルモネラの分離法をご紹介-ビオチンとストレプトアビジン菌をコーティングすることによりマクロファージからファゴゾームを含みます。

サルモネラの分離-大食細胞のファゴゾームを含む

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

isolation-salmonella-typhimurium-containing-phagosomes-from

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

10.5K ビュー.  University of Cologne. 簡単で迅速なサルモネラの分離法をご紹介-ビオチンとストレプトアビジン菌をコーティングすることによりマクロファージからファゴゾームを含みます。

ビデオ: Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

マウスの腸からの樹状細胞およびマクロファージの単離とキャラクタリゼーション

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

免疫・感染学

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

使用<em&gt;フレクスナー赤痢菌</em&gt;オートファジー細胞骨格の相互作用を研究するために

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

表現型のハイスループットアッセイ<em&gt;サルモネラ菌</em&gt;ネズミチフス菌協会、侵略、およびマクロファージにおける複製

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

液胞対細胞質の定量化<em&gt;サルモネラ</em&gt;差動透過化初代マクロファージで

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

フローサイトメトリーによるラットの腎臓からのマクロファージの表現型の特徴

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

レジオネラ・ニューモ外膜小胞：マクロファージ上における炎症誘発ポテンシャルの単離と解析

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

肥満に関連する炎症によって影響を受ける組織のマクロファージの精製と性質、分離

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

離とマウスとヒトの肺胞マクロファージの体外培養

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

慢性サルモネラ感染誘発性腸線維症

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key players in health and disease, acting as phagocytes during immune defense and mediating trophic, maintenance, and repair functions. Although it has been possible to study some of the molecular processes involved in human macrophage function, it has proved difficult to apply genetic engineering techniques to primary human macrophages. This has significantly hampered our ability to interrogate the complex genetic pathways involved in macrophage biology and to generate models for specific disease states. An off-the-shelf source of human macrophages that is amenable to the vast arsenal of genetic manipulation techniques would, therefore, provide a valuable tool in this field. We present an optimized protocol that allows for the generation of macrophages from human induced pluripotent stem cells (iPSCs) in vitro. These iPSC-derived macrophages (iPSC-DMs) express human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169, and our live-cell imaging functional assay demonstrates that they exhibit robust phagocytic activity. Cultured iPSC-DMs can be activated to different&#160;macrophage states that display altered gene expression and phagocytic activity by the addition of LPS and IFNg, IL4, or IL10. Thus, this system provides a platform to generate human macrophages carrying genetic alterations that model specific human disease and a source of cells for drug screening or cell therapy to treat these diseases.

This protocol describes the robust generation of macrophages from human induced pluripotent stem cells, and methods for their subsequent characterization. Cell surface marker expression, gene expression, and functional assays are used to assess the phenotype and function of these iPSC-derived macrophages.

多能性幹細胞からのヒトマクロファージの生成と特性

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key ...

サルモネラの分離-大食細胞のファゴゾームを含む

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