この研究は国立衛生研究所からの助成金とテキサス大学アーリントン校MMに資金をセットアップすることによってサポートされていました。

1。成長する植物と準備細菌<ol><li> V：：Vの生育培地の混合物（熱地-地球のプラグと苗ミックス、サングロ）、細かいバーミキュライト、パーライトおよびこの手順を開始するには、午前1時01分01秒Vのシロイヌナズナの種子をまく。 </li><li>成長チャンバー（22℃、100μモル/ mm 2の /秒毎日の光と65の12時間± 5％湿度）及び必要に応じて水で植物を育てる。植物は、彼らが前にボルトに若い完全に展開された葉を持っている場合4-6週間で使用する準備が整いました。 </li><li>実験を始める前に二日は、細菌の培養を準備。修正されたLB培地（10g / Lのトリプトン、5g / Lの酵母エキス、5g / lの塩化ナトリウム液（pH7.0）、および1.5％寒天）と28で24時間インキュベート℃の上にグ ​​リセロールストックからストリークシュードモナスシリン接種を準備し、常に細菌が継代培養後の弱毒になるとグリセロールストックから培養プレートを開始するために新鮮な培養を使用してください。 </li><li> P. 10 mLの液体培養を開始するためにプレート上に成長した細菌を使用してください50 mLの三角フラスコにシリン 。 28で一晩培養液を振とうしながら精力的で、Cをそれが0.8と1の間の600 nmにおける光学密度またはODに達するまで。 </li><li> 600 ODを測定し、10分間1360 × gで遠心分離により細胞を収穫。最後のODが0.2になるように蒸留水で細胞を再懸濁します。このODは10 8コロニー形成単位（CFU）/ mLに相当する。準備が細菌で、葉を準備し、アッセイを行うために進んでください。 </li></ol> 2。バクテリアと葉の染色とインキュベーション<ol><li>染色するために、葉はまず水に20μMのヨウ化プロピジウムまたはPI溶液を調製。溶液10 mL、一度に3つの小さな葉を染色するのに十分です。 </li><li>成長チャンバーから植物を取得し、鉗子で3若い、完全に展開した葉を切り離します。 5分間のPI溶液中で全体の葉を浸し。その後、葉を取り除き、蒸留水で簡単にそれらを洗い流してください。 </li><li>次の場所下面は、顕微鏡スライド上に下向きに持つ葉。葉がスライド上に平らになるようにミッド静脈を除く4つの部分に鋭いカミソリの刃を持つ葉を切り取ります。 </li><li>葉で細菌を培養するために、スライド上の葉の部分の下に細菌懸濁液300μLを加える。葉の下面は、接種に接触していることを確認してください。 </li><li>植物を育てるために使用されたのと同じ環境条件でサンプルをインキュベートする。顕微鏡下で葉を観察する時に、上を向く下面との新しいスライドに葉の部分を転送する。スライドをマウントするときに、カバースリップが使用されていないことに注意してください。同じ葉の試料はインキュベーションの間に複数回撮像することができ、時間の経過は、研究目的に応じて設定することができる。 </li></ol> 3。顕微鏡、測定とデータ解析<ol><li>ここでは共焦点顕微鏡（LSM 510メタ、カールツァイス社）をスキャンするレーザーは、葉を調べるために使用されます。 PI（励起453 nmの発光543および620nm）の蛍光を検出するために、葉の下面を観察。 </li><li>同じ葉のサンプルを使用すると、時間の経過とともに気孔の画像をキャプチャ。気孔開口部の幅を測定するために画像を保存します。 </li><li> LSMの画像のブラウザを使用して、各時点における各治療のために少なくとも60気孔の気孔開口部の幅を測定します。 </li><li>気孔開口部の幅の平均と標準誤差を計算する。結果の統計的有意性は、2尾、ペアワイズスチューデントのt検定を用いて計算することができます。 </li></ol> 4。細菌とのインキュベーションに気孔応答の代表的な画像<ol><li>ここで明視野のビュー（図1）におけるレーザー走査型共焦点顕微鏡下でシロイヌナズナの葉の表面の典型的な顕微鏡画像は、（20倍対物レンズを用いて）です。 </li><li>ヨウ化プロピジウム染色生細胞の細胞壁は、データの収集（図2）のための無傷の細胞の同定を可能に加えて、孔辺細胞の可視性を高める。気孔孔開口部の範囲が広く開いている気孔に完全に閉じた気孔から、これらの顕微鏡写真で見ることができます。 </li><li>完全に閉じた気孔は孔辺細胞の形状（図3）によって識別されます。開口幅は、0μmと考えられている。 </li><li>オープン気孔（図4）で示した開口幅は、気孔孔の広い領域にわたって描かれた直線を用いて測定されます。 </li><li>これらは、この気孔アッセイの代表的な実験結果です。Arabidopisの葉は、 シュードモナスシリンの3つの異なる株で、暗所でインキュベートした。唯一の細菌は、PST DC3000は、菌株ごとにランダムに選ばれた60気孔（図5）の開口幅で測定された暗閉じた気孔を、開くことができました。 </li></oL&gt; <img src="/files/ftp_upload/2185/2185fig1.jpg" alt="図1" /> 図1。 シロイヌナズナの葉の表面の顕微鏡写真。20倍の対物レンズを用いてレーザー走査型共焦点顕微鏡（LSCM）下葉の表面のビューの1つのフィールドを変更します。気孔の開口は、ヨウ化プロピジウム染色（図2）によって支援蛍光ビューと比較して明らかなようにではないことに注意してください。 <img src="/files/ftp_upload/2185/2185fig2.jpg" alt="図2" /> 図2。ヨウ化プロピジウム染色シロイヌナズナの葉の表面の顕微鏡写真。20倍の対物レンズを用いてレーザー走査型共焦点顕微鏡（LSCM）下葉の表面のビューの1つのフィールド。気孔の孔の開口部の範囲に注意してください。黄色の矢印は、完全に気孔を閉じるために指摘されていると緑色の矢印が広く開いた気孔を指摘されています。 <img src="/files/ftp_upload/2185/2185fig3.jpg" alt="図3" /> 図3。の形状と孔辺細胞の間の開口部で識別される、完全に近い気孔。開口幅は、0μmと考えられている。 <img src="/files/ftp_upload/2185/2185fig4.jpg" alt="図4" /> 図4。ミクロンの開口幅を示す開いた気孔測定は、気孔の孔の広い領域にわたって引かれた直線に基づいてLSCMブラウザを使用して撮影した。 <img src="/files/ftp_upload/2185/2185fig5.jpg" alt="図5" /> 図5。 シュードモナスシリン PVの3株とインキュベートしたシロイヌナズナの葉の気孔開口トマト ：。。DC3118、DB29、およびDC3000植物は、実験時に暗闇に格納されていました。結果は平均値（N = 50から70まで）±標準誤差として示されています。統計学的有意性は、両側スチューデントt -テストで検出した。実験は、同様の結果を3回繰り返した。

<ol>
	<li>Brooks, D. M., Hernandez-Guzman, G., Kloek, A. P., Alarcon-Chaidez, F., Sreedharan, A., Rangaswamy, V., Penaloza-Vazquez, A., Bender, C. L., Kunkel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+well-defined+series+of+coronatine+biosynthetic+mutants+of+Pseudomonas+syringae+pv.+tomato+DC3000.">Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonas syringae pv. tomato DC3000.</a> Mol Plant-Microbe Interact. 17, 162-174 (2004).</li><li>Katagiri, F., Thilmony, R., He, S. Y., Somerville, C. R., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+thaliana-Pseudomonas+syringae+interaction.">The Arabidopsis thaliana-Pseudomonas syringae interaction.</a> The Arabidopsis Book. , (2002).</li><li>Ma, S. W., Morris, V. L., Cuppels, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+DNA+region+required+for+production+of+the+phytotoxin+coronatine+by+Pseudomonas+syringae+pv.+tomato.">Characterization of a DNA region required for production of the phytotoxin coronatine by Pseudomonas syringae pv. tomato.</a> Mol Plant-Microbe Interact. 4, 69-74 (1991).</li><li>Melotto, M., Underwood, W., Koczan, J., Nomura, K., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+stomata+function+in+innate+immunity+against+bacterial+invasion.">Plant stomata function in innate immunity against bacterial invasion.</a> Cell. 126, 969-980 (2006).</li></ol>

利害の衝突は宣言されません。

我々は異なる治療に気孔応答の簡単な評価を可能に無傷の葉組織に気孔開口部を測定するためにまっすぐ進むの手順を提示している。 私たちはモデルシステムのシロイヌナズナ / シュードモナスシリンを使用して結果を提示されているが、無傷の葉気孔のアッセイは、潜在的に任意の植物-細菌の組み合わせで行うことができます。プロトコルは、簡単に他の植物や細菌の病原体の成長の必要条件に合わせて変更することができます。全体的な原則と手順は同じです。さらに、このメソッドは機能的な孔辺細胞の出力だけでなく、微生物を生きるために勉強したい研究者に有益なだけでなく、葉の自然環境を維持する条件下で、他の刺激と化学物質への可能性があります。 全体の葉は、その厚さや不均一な地形のため、画像に困難になる可能性があります。この問題は、スライド上に平らように葉の中央静脈を除去し、共焦点顕微鏡を使用することによって軽減することができます。しかし、蛍光顕微鏡の他のタイプは、葉の表面の高解像度の画像を取得するために使用することができます。

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<td>Propidium iodide</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P4864</td>
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<td>Tryptone</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP1421-1</td>
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<td>Yeast Extract</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>0127-01-7</td>
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<td>Sodium Chloride</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>7647-14-5</td>
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<td>Agar</td>
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<td>J637-2500G</td>
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<td>Plant growth chamber</td>
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<td>Shaker incubator</td>
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<td>Laser scanning confocal microscope</td>
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assessing stomatal response to live bacterial cells using whole leaf imaging

気孔は、植物のインテリアと環境の間でガス交換を担当し、植物の表皮の自然な開口部です。彼らは光強度、二酸化炭素濃度、および相対湿度（RH）などの外部要因の数に応じて気孔気孔を閉じたりしている孔辺細胞、のペアによって形成される。気孔の孔は、また、病気の開発のための重要なステップの葉への病原体のエントリのための主要な経路である。最近の研究では、細孔の閉鎖は、 シロイヌナズナ植物に細菌性疾患の開発を最小限に抑えるのに有効であることを明らかにした。植物自然免疫の重要な部分を。以前は、我々は細菌を（。Melottoら、2006）生きて気孔の応答を評価するために表皮の皮を使用しているが、植物表皮のピーリングと細菌の細胞の両方のために有利な環境条件を維持するには挑戦されています。葉の表皮は、孔辺細胞の電気生理学的実験のためのMES緩衝液（10mMのKCl、25mMのMES - KOH、pHは6.15）との生存と健康的に保つことができます。しかし、このバッファは、細菌懸濁液を得るためには適切ではありません。一方、細菌細胞は、倍の長時間表皮皮を維持するために、適切ではない水に保つ事が可能。水で表皮剥離フロートは、4時間以内に空気乾燥にさらされている皮の細胞は、実験を実施するタイミングを制限するとき。孔辺細胞の特定の刺激の効果を評価するための理想的な方法は、気孔生理学にし、できるだけ多くの植物の自然環境への干渉を最小限に提示する必要があります。我々は、したがって、葉の傷や操作が大幅に容易に再現性と信頼性の高い気孔アッセイを提供することを目指して最小化されている細菌を生きて気孔の応答を評価する新しい方法を開発した。プロトコルは、ヨウ化プロピジウム（PI）、細菌懸濁液を葉の染色のインキュベーション、および共焦点顕微鏡を走査型レーザーの下での葉の観察と生葉の染色に基づいています。最後に、このメソッドは、密接に、植物が病原体によって攻撃される際の自然条件を模倣する条件を使用して、長時間にわたって同じライブ葉の試料の観察が可能になります。

Watch this Scientific Journal Video about Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging at JoVE.com

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

我々は、生菌に対する気孔の応答にアクセスするためのシンプルで再現性のプロトコルを開発しました。表皮皮の使用と比較して、この方法では、葉の傷や操作を最小限に抑えることが以前に報告した。

全葉のイメージングを使用して細菌細胞をライブに気孔反応を評価する

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

assessing-stomatal-response-to-live-bacterial-cells-using-whole-leaf

Research

JoVE Journal

Immunology and Infection

18.2K ビュー.  University of Texas at Arlington. 我々は、生菌に対する気孔の応答にアクセスするためのシンプルで再現性のプロトコルを開発しました。表皮皮の使用と比較して、この方法では、葉の傷や操作を最小限に抑えることが以前に報告した。

ビデオ: Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

マウスでイメージ細菌感染にルシフェラーゼを用いた

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

免疫・感染学

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 ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

生物発光細菌のイメージング<emインビボで&gt;</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

光活性化単一分子追跡を使用してライブ細菌細胞におけるタンパク質-DNA相互作用を可視化する

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

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

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

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

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

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

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

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

使用した植物防御応答のファインキャラクタリゼーションのための細菌の葉浸潤アッセイ<em&gt;シロイヌナズナシュードモナスシリン</em&gt; Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

の生理反応を測定します<em&gt;ショウジョウバエ</em&gt;ライブカルシウムイメージングを使用して脂質フェロモンの感覚ニューロン

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

に応答したヒト一次好中球及び単球による抗カビ活動のライブイメージング<em&gt; A。フミガーツス</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

ミバエショウジョウバエVivo で貪食能アッセイを使用しての細胞性免疫能を評価します。

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

正確な細胞表現と正確なタンパク質局在化のための細菌細胞の三次元イメージング

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...