私たちは、原稿の重要な読書のためのファゴソームの生物学研究室のメンバーに感謝。この作品は、ヘルムホルツ若手研究助成（イニシアティブやヘルムホルツ協会のネットワーク化ファンド） と優先プログラムドイツ研究協会（ドイツ学術振興、DFG）のSPP1580グラントによってサポートされています。

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+phagocytic+engulfment+of+apoptotic+cells+by+macrophages+using+pHrodo+succinimidyl+ester.">Measurement of phagocytic engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester.</a> Current protocols in immunology. 14, (2013).</li><li>Rogers, L., Foster, L. <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> Proc. Natl. Acad. Sci. U.S.A. 104, 18520-18525 (2007).</li><li>Russell, D. G., Vanderven, B. C., Glennie, S., Mwandumba, H., Heyderman, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+macrophage+marches+on+its+phagosome:+dynamic+assays+of+phagosome+function.">The macrophage marches on its phagosome: dynamic assays of phagosome function.</a> Nat. Rev. Immunol. 9, 594-600 (2009).</li><li>Savina, A., Vargas, P., Guermonprez, P., Lennon, A. -. M., Amigorena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+pH,+ROS+production,+maturation,+and+degradation+in+dendritic+cell+phagosomes+using+cytofluorometry-based+assays.">Measuring pH, ROS production, maturation, and degradation in dendritic cell phagosomes using cytofluorometry-based assays.</a> Methods. Mol. Biol. 595, 383-402 (2010).</li></ol>

著者らは、開示することは何もありません。

次のセクションでは、提示された方法とその限界の重要なステップを説明します。さらに、我々は、その解決策の一般的な問題のいくつかを接続可能な修飾を導入し、本手法の利点だけでなく、このアプローチに適し補完的な方法を検討していきます。 ビーズ表面へのIgGの結合を含むビーズの調製は、重要であり、unfresh製剤の使用は避けるべきである。それへの追加で?...

マクロファージを含む専門的な食細胞は、免疫系で重要なを果たしている。先天性免疫系における最初の防衛線であることに加えて、それらはまた、それらのシグナル伝達およびロール1-3抗原提示を介して適応免疫の活性化において重要な役割を果たす。専門的な食細胞の機能は多面的ですが、ファゴソームの成熟は、プロの食細胞4,5の殺菌及び抗原処理機能の重要なバックボーンです。受容体を介した貪食や細菌などの貪食ターゲットの取り込み時には、発生期のファゴソームは、相互作用し、エンドサイトーシスネットワークのコンパートメントおよび他のいくつかの細胞小器官6の交流の複雑でよく編成シーケンスを通過します。化合物の性質は、これらの細胞の実体だけでなく、規制と交換し、融合事象のタイミングは、管腔ファゴソーム環境とphagosを決定OMALの膜組成と、このように7、成熟ファゴソーム8の運命。 ファゴソーム成熟の生理的関連性は、から逃げる停止またはファゴソーム成熟9を破壊するために、様々な細胞内病原体によって使用される多様な戦略によって例示される。これらの戦略の最も直接的または間接的にファゴソームの成熟過程の最終的かつ重要な段階を防ぐ：加水分解酵素と成熟ファゴソーム7の抗菌要因の大部分とそれらを賦与後期エンドソーム/リソソームコンパートメントファゴソームの融合、 、8,10。この最終的な重要なステップの分析は、したがって、ファゴソームの成熟の状態について、自然な生理的な状態は、正または負に利用されている特定の実験設定で影響を受けているかの強力な指標を提供してくれることができます。 後期エンドソーム/リソソーム区画sが、一般に種々のステージ特異的マーカー分子の存在により早期エンドサイトーシス区画から、エンドサイトーシス経路の末端区画とみなす定義され、区別される。例えばこのような他の人の間で11リソソーム関連膜タンパク質（ランプ）などの加水分解酵素や膜成分のため。ターミナルエンドサイトーシスは、区画-今後リソソームは、また、食細胞/エンドサイトーシス経路12の最後に消化の最終段階のための主要な場所として機能し、単にと呼ばれる。このように、難消化性プローブは、細胞外環境からのエンドサイトーシスおよびマクロピノによってロードすることができ、それを取り込み、チェイスの定義されたサイクルに従った後13,14を蓄積リソソームへのエンドサイトーシス経路を通って輸送される。 15-17 endocyticprobesなどの有機難消化性高分子デキストランのような蛍光顕微鏡用の蛍光団結合プローブは、一般的に使用されている。 <p clasこの方法ではs = &quot;jove_content&quot;&gt;、我々は、ファゴソーム、リソソーム内腔にコンテンツの配信を分析することによって、ファゴソーム成熟を研究する貪食カーゴとしてIgGをコーティングしたマイクロビーズの使用を記載している。ライブ骨髄由来マクロファージ（BMM）、共焦点レーザー走査顕微鏡でタイムラプスビデオ画像内のリソソームの容易に検出可能なマーカーとして蛍光団結合デキストランプローブを用いて、我々は高とファゴソームへの貨物輸送の動的なプロセスに従う時間分解能。次に、フィジー（又はImageJの）収集タイムラプス撮影データは、オープンソース、無料利用可能な画像解析ソフトウェアを用いて評価することができる方法を説明し、統計的にMicrosoft Excelを用いて解析し、グラフパッドプリズムソフトウェア14,18を用いて提示した。 我々の方法の説明に続いて、類似の実験は、ファゴソーム成熟に対するそれらの影響を調査するために変更された設定および因子を使用して設計することができ、実質的に任意のオト付着性の一次または細胞ラインの貪食細胞の彼女のタイプ、遺伝子ノックアウトやノックダウンを、変異体または融合タンパク質の発現、食細胞 - ターゲット、マイクロビーズコーティング化合物、エンドソーム/リソソームプローブや要因などAを含む機能実験の遺伝的損失とりわけ、サイトカイン、化学阻害剤、またはsiRNAは、この方法の基本的なアプローチを適用することができるすべての変数である。 私たちは、このメソッドは次のようにするという目標や基本的な要件を定義します。 <ul type="disc"><li>この方法の目標：食細胞を生きた高い時間分解能でファゴソームの成熟の、直接的な定量的および定性的観察と分析を可能にする。 </li><li>貪食貨物：適切にコーティングされた微粒子または生物学的標的。ここでは、容易に食作用を媒介するのFc-γ受容体を介して内在化さIgGをコーティングした3μmの球状のポリスチレン微粒子を使用しています。代わりに、任意の微生物またはコーティングされたMIC貪食受容体が細胞上に存在するためにroparticleを使用することができる。 </li><li>食細胞：適切な貪食受容体を発現する接着食細胞。ここでは、豊富のFc-γ受容体を発現するマウス初代BMMSを使用しています。あるいは、樹状細胞（DC）、単球または食作用上皮細胞またはそれらの遺伝的変異体またはノックアウト変異体を使用することができる。 </li><li>リソソーム貨物：蛍光標識したリソソームプローブ。ここで、テキサスレッドコンジュゲート7万キロダルトンのデキストラン（Dex70kD）はリソソームの管カーゴとして使用される。あるいは、そのような酸味や、分解能力などのファゴソームプロパティの細胞区画または細胞小器官、または適切なプローブのいずれかの蛍光検出可能なマーカーは、ファゴソーム成熟の進行を研究するために使用することができた。 </li><li>例えば分子、化学化合物、遺伝子ノックダウン、または変異タンパク質の一過性発現を可能性があり、シグナリング：要因に影響を与える。ここでは、FORGを持っている簡単のためにこのような因子の1使用していますが、変異型タンパク質の発現およびKasmapour ら 18ファゴソームの貪食や状況と移動性に影響を与えると/できるあらゆる要因を参照してくださいノックダウンに影響を与える要因との最近の例または成熟ファゴソームと対話コンパートメントは、潜在的に使用することができる。 </li></ul>

<table border="0" cellpadding="0" cellspacing="0" width="610">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagles Medium (D-MEM)</td>
			<td>PAA, Austria</td>
			<td>E15-009</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagles Medium without phenol red (D-MEM)</td>
			<td>PAA, Austria</td>
			<td>E15-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>PAA, Austria</td>
			<td>A15-151</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>PAA, Austria</td>
			<td>M11-004</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>PAA, Austria</td>
			<td>H15-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>PAA, Austria</td>
			<td>P11-010</td>
			<td></td>
		</tr>
		<tr>
			<td>WillCo-dish&#174; Glass bottom dish (35 mm &#8709;, #1.5)</td>
			<td>WillCo Wells, Netherlands</td>
			<td>GWSt-3522</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLviewTM glass bottom dish, 35 mm</td>
			<td>Greiner Bio one, Germany</td>
			<td>627871</td>
			<td>Alternative: Available as segmented</td>
		</tr>
		<tr>
			<td>Carboxylated latex microspheres</td>
			<td>Polysciences, USA</td>
			<td>#09850</td>
			<td>Available in different diameters, we used 3 &#956;m</td>
		</tr>
		<tr>
			<td>Polystyrene microparticles</td>
			<td>Kisker Biotech, Germany</td>
			<td>PPs-3.0COOH</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>ROTH, Germany</td>
			<td>305.1.3</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse whole IgG</td>
			<td>Rockland, USA</td>
			<td>010-0102</td>
			<td></td>
		</tr>
		<tr>
			<td>EDAC crosslinker</td>
			<td>Sigma-Aldrich, USA</td>
			<td>E6383-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM Tris</td>
			<td>Sigma-Aldrich,USA</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>1-ml syringe Omnifix -F</td>
			<td>B.Braun, Germany</td>
			<td>9161406V</td>
			<td></td>
		</tr>
		<tr>
			<td>26&#160;G needle (0.45*25 mm)</td>
			<td>B.Baun, Germany</td>
			<td>4657683</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>PAA, Austria</td>
			<td>E15-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Gibco, USA</td>
			<td>16050-122</td>
			<td></td>
		</tr>
		<tr>
			<td>MES hydrate</td>
			<td>Sigma-Aldrich, USA</td>
			<td>M2933</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 &#956;M syringe mounted filter</td>
			<td>Sartorius, Germany</td>
			<td>83.1826.001</td>
			<td></td>
		</tr>
	</tbody>
</table>

study of phagolysosome biogenesis in live macrophages

貪食細胞はそのファゴソーム内侵入する微生物を除去して除去することにより、自然免疫系において主要な役割を果たしている。ファゴソームの成熟は、新生ファゴソームが細胞質内の種々の細胞小器官とコンパートメントよく編成の相互作用を介して抜本的な転換を受けている間、複雑で厳密に調節されたプロセスである。その溶解性と殺菌性とファゴソームを持たせることにより、食細胞の生理機能に必須であるこのプロセスは、リソソームとファゴソームのための成熟の最後と重要な段階であると考えられているファゴリソソームの生合成とファゴソームの融合で絶頂に達する。本稿では、ファゴリソソーム生合成の特徴であるコンテンツ配信を、ファゴソームするリソソームの動的な過程の定性的および定量的分析のための生細胞イメージングベースの方法を説明します。このアプローチは、phagocyのモデルとしてのIgGでコーティングされたマイクロビーズを使用しています管腔リソソーム貨物プローブとしてtosisと蛍光団結合デキストラン分子、タイムラプスイメージング、共焦点レーザー走査顕微鏡を使用してライブマクロファージにおけるリアルタイムにファゴソームにlysosmalコンテンツの動的配信を追跡するために。ここでは、準備手順や他のラボで、この方法の簡単で正確な展開を可能にするためのステップバイステップの実験、詳細に背景を説明します。我々の記載された方法は、単純な堅牢な、そして最も重要なことは、容易に異なるシステムや様々な食細胞タイプ、機能喪失実験を、異なるプローブの使用などの様々な実験設定でファゴソームの相互作用および成熟を研究するために適合させることができるであり、そして貪食粒子。

イメージングのための細胞やビーズの正しい準備が非常に重要です。 図1に、BMMSための私達の最適化されたプロトコルに基づいて播種、プローブのロードと、この方法では、最初の画像化手順の概要を示している。これは、プロトコル·パラメータが使用される細胞およびプローブの種類に応じてテストし、最適化することが不可欠である。 プリロードされ?...

Watch this Scientific Journal Video about Study of Phagolysosome Biogenesis in Live Macrophages at JoVE.com

Study of Phagolysosome Biogenesis in Live Macrophages

ライブマクロファージにおけるファゴリソソーム生合成の研究

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

study-of-phagolysosome-biogenesis-in-live-macrophages

Research

JoVE Journal

Immunology and Infection

14.3K ビュー.  Helmholtz Centre for Infection Research. ライブマクロファージにおけるファゴリソソーム生合成の研究

ビデオ: Study of Phagolysosome Biogenesis in Live Macrophages

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

動的な生細胞イメージングのための宿主 - 病原体相互作用の研究のための光トラップの使用

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

免疫・感染学

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

マウスのマクロファージの枯渇と再構成

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

設立<em&gt;インビトロ</em&gt;システムの細胞内挙動を研究するために、<em&gt;カンジダ·グラブラタ</em&gt;人間のTHP-1マクロファージにおける

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

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 ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

2D電気泳動によるマクロファージのプロテオームプロファイリング

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

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 ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

HIV-1に感染したライブ初代ヒトマクロファージで測定ファゴソームの移行と速度

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

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 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 ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

FcγR 架橋への応答におけるマクロファージの活性酸素産生の流れフローサイト メーターによる測定

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...