この研究は、健康研究 (機構) 営業許可モップ-123419、自然科学と工学研究会のカナダ発見グラント 418194 とオンタリオ州省の研究、BH に初期研究賞のカナダの協会によって賄われていた。DGW はいくつかの画像を提示、プロトコルの最適化と; 原稿の執筆に貢献してください。彼はリヴァプールの大学からエコカー補助金で賄われていた。CY はヴァニエ大学院奨学金とされて MD/思い立ったによって資金を供給します。資金提供機関は、研究デザイン、データ収集と分析、意思決定を発行し、または原稿の準備で役割をなかった。

著者は利益相反を宣言しません。

このプロトコルで説明する方法では、イメージング、固定細胞と生細胞の両方の方法を使用して、動的な efferocytic プロセスの定量化を有効にします。これらのアプローチは、一般的に使用される流れ cytometry 法23,24上のいくつかの利点を提供しています。インサイド アウト染色固定サンプルの使用率のより堅牢で正確な定量化と efferocytosis の範囲?...

アポトーシスまたはプログラム細胞死は、ほとんどの多細胞生物の発生、発達と恒常性1の重要な非常に調整される生理学的プロセスです。正常細胞の代謝回転と萌芽期の開発に関与している、に加えてアポトーシス組織から感染したり、損傷した細胞の除去を有効にし、感染、炎症、癌とも医療介入による応答で引き起こされることができます。放射線療法やステロイド1。アポトーシス細胞を公開"を食べる-私"専門家と非専門家の食細胞の範囲の受容体によって認識される表面に信号をまとめて"efferocytes"と呼びます。これらの受容体の関与は、efferocytosis2,3として知られているプロセスによって efferocyte によって吸収とアポトーシス細胞の劣化を誘導します。ホスファチジルセリンは最高の特徴を食べる-運転 efferocytosis シグナルします。それは通常アポトーシス細胞表面4ホスファチジルセリンを公開するので、この膜非対称を乱す脂質 scramblase をアクティブにすると、血しょう膜の内部のリーフレットに限定されます。ホスファチジルセリンは、成熟マクロファージなどのいくつかの非アポトーシス細胞の細胞表面にあるし、血小板を活性化します。しかし、これらの細胞は、その細胞表面5,6,7CD47 などの「私を食べていない」の信号の存在のために efferocytosed ではありません。露出ホスファチジルセリンは、efferocytes で表される efferocytic 受容体の配列によって認識されます。ホスファチジルセリンにこれらの受容器のバインディング、直接または、オプソニンの援助は efferosome8,と呼ばれる膜結合液胞にアポトーシス細胞の貪食を促進するシグナル伝達経路をアクティブに9,10,11,12.、efferosome は順番にエンドソームと融合し、貨物13,14細胞のリソソームは、分子機械、efferosome を酸性化して、アポトーシスを低下する必要を提供します。リサイクルのエンドソーム アポトーシス細胞由来材料劣化、一度入稿-アポトーシス細胞由来の抗原に対する免疫応答を制限して、可能性がありますによりアポトーシス細胞13から栄養素の回復プロセス 15。Efferocytosis で故障した場合、アポトーシス細胞の障害クリアランスこれらの未確認の細胞は最終的に二次壊死を受けます。壊死細胞は、感染、炎症、自己免疫疾患16,17の範囲を駆動、細胞外の環境に賛成して炎症性のゾル性細胞質の内容、病原体・自己抗原をリリースします。一緒に、アポトーシスと efferocytosis 死んで、死んだ細胞の除去を容易にするため、組織の恒常性の維持を可能にします。
Efferocytosis の基礎となる分子機構を解明、アポトーシス細胞取り込みの明確な数量を提供するメソッドが必要です。他の吸収とは異なりエンドサイトーシスと貪食18,19、efferocytosis などのメカニズムがない結果は断片的な吸収の結果そのままターゲット細胞の貪食で、この数量は複雑します。efferocyte20によるアポトーシス細胞。記載プロトコルの in vitro efferocytosis 試金を記述、内面の正確な描写を提供する個々 のアポトーシス細胞の非内面部分とさまざまな固定セルと組み合わせることができますと生細胞の顕微鏡に近づきます。伝統的な食作用の試金の抗体を追加手法として内在化している非ターゲットをラベル付けするために実験の終わりに貪食のターゲットに固有の共有リンク ビオチン21アポトーシス ターゲットのラベルによって異なります,22. ビオチン化アプローチに分類されるべき任意のタンパク質含有ターゲットを可能にし免疫染色を行った場合、二次抗体の交差反応性の潜在的な問題を回避アポトーシス細胞特異的抗体は、この試金で使用できますが、.具体的には、我々 は、アポトーシス Jurkat 細胞追跡の染料とビオチンの両方でデュアル染色されているの準備を概説します。追跡色素細胞はアポトーシス細胞由来材料 efferocytosis 中に追跡する表面ビオチン化可能の差別に対し efferocytosed アポトーシス細胞の非内面部分から内面のことができます。文化とマウスと人間の efferocytes として使用、M2 マクロファージの J774.2 と THP 1 細胞の efferocytic ターゲットとして一次電池 efferocytosis および Jurkat 細胞の例として準備について述べる。これらのメソッドは、他の細胞や細胞、脂質コーティングを介してアポトーシスをシミュレートするミクロン サイズの模倣し、標的細胞の細胞死 (アポトーシスなど壊死とネクロトーシス) の任意のフォームを受けているに簡単に適用できるか特定の関心の efferocytic 受容体にリガンドでコーティングします。
このプロトコルで説明したメソッドには、フィールド23,24でよく使用される流れ cytometry による方法に比べていくつかの利点があります。両方の合計と内在化している非アポトーシス細胞材料の明確なラベリングと組み合わせる食細胞アポトーシス細胞の相互作用を直接イメージングによる efferocytosis の定量的な対策が可能です。また、pH に依存しない fluorophores の使用は、いくつかの代替方法25を混同するライソゾームの pH で FITC と GFP の蛍光性の抑制などの交絡因子を制限します。最後に、詳細に記述されていませんが、これらのメソッド使用できる蛍光標識遺伝子を表現する efferocytes を使用してまたはポスト固定免疫染色、シグナリング分子の活動との監視の定量化を可能にするのにはefferocytosis 中細胞プロセスは。

<table>
	<tbody>
		<tr>
			<td>RPMI 1640 Media</td>
			<td>Wisent</td>
			<td>3500-000-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Media</td>
			<td>Wisent</td>
			<td>319-005-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Wisent</td>
			<td>080-150</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Wisent</td>
			<td>311-010-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>18 mm circular glass coverslips #1.5 thickness</td>
			<td>Electron Microscopy Sciences</td>
			<td>72290-08</td>
			<td>Size and shape of coverslip is not critical, but 18 mm fit into the wells of a standard 12-well plate which simplifies cell culture</td>
		</tr>
		<tr>
			<td>Staurosporine</td>
			<td>Cayman Chemical</td>
			<td>81590</td>
			<td>Dissolve in DMSO at 1 mM (1,000x stock solution)</td>
		</tr>
		<tr>
			<td>Annexin V-Alexa 488</td>
			<td>ThermoFisher</td>
			<td>R37174</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-Link NHS-Biotin</td>
			<td>ThermoFisher</td>
			<td>20217</td>
			<td>Store in a dessicator. Do not prepare a stock solution.</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace FarRed</td>
			<td>ThermoFisher</td>
			<td>C34572</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace Orange</td>
			<td>ThermoFisher</td>
			<td>C34851</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoescht 33342</td>
			<td>ThermoFisher</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-Streptavadin</td>
			<td>ThermoFisher</td>
			<td>SA1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Lympholyte-poly cell sepration medium</td>
			<td>Cedarlane Labs</td>
			<td>CL5071</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human M-CSF</td>
			<td>Peprotech</td>
			<td>200-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IL-4</td>
			<td>Peprotech</td>
			<td>300-25</td>
			<td></td>
		</tr>
		<tr>
			<td>J774.2 Macrophage Cell Line</td>
			<td>Sigma-Aldrich</td>
			<td>85011428-1VL</td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1 Human Monocyte Cell Line</td>
			<td>ATCC</td>
			<td>TIB-202</td>
			<td></td>
		</tr>
		<tr>
			<td>Jurkat T Cell Line</td>
			<td>ATCC</td>
			<td>TIB-152</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of efferocytosis by single-cell fluorescence microscopy

Efferocytosis の規制を勉強してアポトーシス細胞の取り込みを正確に定量化する efferocytosis を制御するシグナル伝達と細胞プロセスをプローブすることができる方法が必要です。この数量は、アポトーシス細胞は efferocytosed、断片的、呼び出しと残留 unengulfed 細胞に対するアポトーシス ターゲットの efferocytosed 部分の間正確に記述することができます。 メソッドを実行することは困難することができます。フラグメント。記載方法は、正確にマクロファージなどの efferocytes の efferocytosis と efferocytic の容量のダイナ ミックスを定量化するためのアプローチのデュアル ラベルを利用しています。アポトーシス細胞の細胞質がアポトーシス細胞の表面ビオチン化可能ですが差別化と非内面内面の間すべてのアポトーシス細胞由来物質の監視を有効にするための細胞追跡染料にラベル付けされてアポトーシス細胞画分。Efferocytes の efferocytic 容量は、ライブまたは固定細胞の蛍光画像を撮影し、ストレプトアビジンの汚損によって差別化、内面化されたターゲット対連結の量を定量化によって決定されます。このアプローチは、フローサイトメトリー、すなわち efferocytosed アポトーシス細胞画分、生細胞顕微鏡による efferocytic のダイナミクスを測定する能力と非 efferocytosed の正確な描写などの方法と比べていくつかの利点を提供していますと、蛍光標識した遺伝子を発現する細胞の細胞内シグナル伝達の研究を実行する能力。組み合わせることで、このプロトコルで説明する方法は、正確に efferocytic 活動を定量化し、efferocytosis 中細胞のシグナル伝達経路を調査するために使用することができます柔軟な実験的アプローチのための基礎として機能します。

Jurkat 細胞のアポトーシスに 1 μ M スタウロスポリン結果の文化を一晩 > アネキシン V (図 1) を染色で確認することができます細胞の 95%。スタウロスポリンの濃度とスタウロスポリン処置の持続期間は各セル行の最適化が必要であるが、これらの実験のため他の細胞型を使用できます。信頼性の高い検知及び efferocytosis、定量化の > 細胞の 80% は、...

Watch this Scientific Journal Video about Quantification of Efferocytosis by Single-cell Fluorescence Microscopy at JoVE.com

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Efferocytosis、アポトーシス細胞の貪食除去は恒常性を維持するために必要な受容体と認識、キャニスター、アポトーシス細胞の内面化を可能にするシグナル伝達経路によって促進されます。ここで、efferocytosis の定量化と efferocytic シグナル伝達経路の活性の蛍光顕微鏡のプロトコルを提案する.

単一細胞の蛍光顕微鏡による Efferocytosis の定量化

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

quantification-of-efferocytosis-by-single-cell-fluorescence-microscopy

Research

JoVE Journal

Immunology and Infection

12.4K ビュー.  University of Western Ontario. Efferocytosis、アポトーシス細胞の貪食除去は恒常性を維持するために必要な受容体と認識、キャニスター、アポトーシス細胞の内面化を可能にするシグナル伝達経路によって促進されます。ここで、efferocytosis の定量化と efferocytic シグナル伝達経路の活性の蛍光顕微鏡のプロトコルを提案する.

ビデオ: Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

マイクロ波は、透過型電子顕微鏡による植物ウイルス病の迅速診断をアシスト

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

免疫・感染学

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

の単一細胞解析枯草菌</em蛍光顕微鏡とフローサイトメトリーを用いて>バイオフィルム

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

ライブセル蛍光顕微鏡を用いた細菌毒素誘導される応答の可視化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

細胞内病原体によって引き起こされる液胞破裂のシングルセル測定

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

哺乳動物細胞に関連して細菌の生存を決定するための蛍光顕微鏡法

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

共焦点顕微鏡による絶縁型肺胞マクロファージの抗真菌活性を評価する

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

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 ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

レシオメトリック蛍光顕微鏡によってファゴソームのpHを測定します

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

イヌ好中球細胞外トラップリリースの定量化のためのシンプルな蛍光アッセイ

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

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.

単一細胞の蛍光顕微鏡による Efferocytosis の定量化

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