我々は、貴重な議論のため惠淑ああ、ジョアントムモレロとアランCollmer、植物病理学および植物 - 微生物の生物学、コーネル大学、学科に感謝。我々は記事にコメントをジェシーMunkvoldに感謝。資金は、全米科学財団の植物ゲノムのプログラム、受賞番号DBI - 0605059（GBM）によって提供されていました。

パート1：植物の成長と維持アッセイで使用されているタバコbenthamiana植物は、約7週齢にする必要があります。彼らはすべての腋窩枝や花を削除するには少なくとも4-5日間アッセイの前にトリミングする必要があります。それは植物がより管理しやすくするために、すぐに彼らが出現した後、腋窩枝を削除することをお勧めします。 パート2：細菌培養 1日目： <ol><li>アッセイで使用されているすべての菌株に対してプレートまたは培養物は同時に開始されます。 シュードモナス属用。これは、凍結グリセロールストック（表2）から適切な抗生物質を含有KBMプレート上に2誘導と3チャレンジ株、ストリークの細菌が含まれています。細菌少量のプレートの中央に分散させる必要があります。約18〜24時間30℃でインキュベートする。 、30をアグロバクテリウムの場合は、新鮮なプレートから2 mlのLBで液体培養を開始し、250 rpmで一晩成長℃、 </li></ol> 2日目： <ol start="2"><li> 膿の場合は、細菌に液体KBMの150〜200μlのを追加し、滅菌ガラススプレッダーを使用して全体のプレート上にそれらを広げ。バックインキュベーター内でプレートを入れて、それは別の20〜24時間成長させる。良好な成長を示す、次の日プレート上に細菌の芝生があるはずです。 アグロバクテリウムの場合は、、30を20μMのアセトシリンゴンで約5-10 mlのLBで二次培養を開始し、250 rpmで20時間に一晩成長させ℃に</li></ol>パート3：分析のための準備植物 2日目： <ol><li>ある日、アッセイ前に植物が22から24の部屋° C、〜35から40パーセントRHと一定の光に移動する必要があります。 </li><li>太い黒のマーカーを用いて直径で少なくとも1.5 cmの葉上に円をマーク。 2〜4つの円は葉ごとにマークすることができます。円はよく等間隔であり、好ましくは大静脈と交差させない。よく、展開葉を選択してください。タフやタッチに厚いものと葉の下側部分に円をマーキング避ける古い葉または葉を避けてください。 </li></ol>前の実験に一日の円をマークすると、プロトコルに必須ではありません。しかし、それは、アッセイされる植物が多数ある場合は時間を節約し、それが容易PTIの誘導とチャレンジ接種との間の正確なタイミングを達成できるようになります。 パート4：PTIの誘導 3日目： <ol><li> P.蛍光菌とP.ために10mMのMgCl 2でプレートから細胞を収集するプチダ 。 アグロバクテリウムのために、細胞を収集する文化を遠心し、10mMのMgCl 2の + 10mMのMES pHを5.6に再懸濁します。のMgCl 2 - MESソリューションで遠心分離と再懸濁しますを繰り返します。 600nmでの細胞の光学密度（OD）を測定します。表2に記載されている最後の必要な値に5.1mmに調整します。 膿が最終のMgCl 2 - MESソリューションで10mMのMgCl 2及びアグロバクテリウムに再懸濁してください。 25mlの総容積は約40〜50スポットを接種するのに十分です。 </li><li> 1ミリリットル無針注射器を使用して葉上で事前にマークした円にPTIの誘導物質を浸透させる。植物が浸透していた順序と浸潤が開始された正確な時間を書き留めておきます。 </li></ol>パート5：チャレンジ接種<ol><li> P.を準備シリン PV。 トマト DC3000、一部4.1および表2に記載のように挑戦は PST DC3000ΔhopQ1 - 1と PS PV。 コナジラミ 。 </li><li>誘導が行われたとして、七時間デューサの浸潤後、植物の同じ順序でチャレンジ浸潤を実行します。一般的に、最初の接種の円の外周上の点は2回目接種の円の中心として使用することができます。葉の上に複数のスポットが存在する場合、浸潤が重複していないことを確認してください。 </li><li>正確なCFUの数を決定するために適切な抗生物質を含有KBMまたはLBプレート上でアッセイで使用されているすべてのカルチャのプレート連続希釈。 </li></ol>パート6：PTIの内訳についてはスコアリング 5日目： <ol><li> pstファイル DC3000でチャレンジしたスポットのETIによる細胞死の外観を探します。浸潤の重複するエリア内の細胞死は、PTIの内訳を示していると肯定的な表現型として採点してください。 </li></ol> 7日目： <ol start="2"><li> pstファイルDC3000ΔhopQ1 - 1または PS PVで挑戦されたスポットの疾患に起因する細胞死の外観を探します。 コナジラミは、 </em&gt;。パート6.1で説明したのと同じ方法で、肯定的な表現型のスコア。 </li></ol>コントロール植物（PTIのために妥協すべきではないことが）浸透のオーバーラップ領域で細胞死を示すために開始すると、それ以上の観測を行う停止。 パート7：代表的な結果図1は、アッセイP.の結果を示しています。 蛍光菌は、チャレンジとしてデューサと PST DC3000として使用されました。 <img src="/files/ftp_upload/1442/fig1tmb.jpg" alt="図1" border="0" /> 図1。P. 蛍光菌はN.に浸潤したbenthamiana葉（黒丸）は、PTIを誘導するために、7時間後、スポットが PST DC3000（白い丸）に挑戦された。 FLS2のために沈黙の植物は、P.地域でPTIの内訳を示した蛍光菌は 、（）に浸潤などの細胞死が見られた。沈黙されていないコントロールの植物はPTI（B）の誘導に起因する重複領域には細胞死を示さなかった。赤い矢印が不足または浸潤のオーバーラップ領域における細胞死の存在を示している。写真は2日間浸潤した後に採取した。してください<a href="https://www.jove.com/files/ftp_upload/1442/fig1.jpg">ここをクリックして</a>図1の拡大バージョンを参照すること。 <table border="1"><tbody><tr><td> PTIの誘導物質 </td><td> 細胞死誘発の挑戦 </td><td> 細胞死の性質 </td><td> コードは </td></tr><tr><td> 蛍光菌 55 </td><td> シュードモナスシリン PV トマト（PST。）DC3000 6 </td><td> ETI </td><td> PF / DC </td></tr><tr><td> P.プチダ KT2440 </td><td> pstファイル DC3000 </td><td> ETI </td><td> PP / DC </td></tr><tr><td> P.蛍光菌 55 </td><td> pstファイルDC3000ΔhopQ1 - 1 6 </td><td>病気</td><td> Pf/Q1-1 </td></tr><tr><td> アグロバクテリウムツメファシエンス GV2260 </td><td> P.シリン PV。タバコ11528R </td><td>病気</td><td>アグロ/ Ptab </td></tr></tbody></table> 表1：PTIの誘導とPTIのための細胞死アッセイで使用される細胞死-引き出す微生物の組み合わせ。 <table border="1"><tbody><tr><td> 細菌株 </td><td> 選択培地 </td><td> 最後のODは、実験で使用 </td><td> 対応するCFU / mlの </td></tr><tr><td> P.蛍光菌 55 </td><td> KBMアンピシリン（100 ug / mlと） </td><td> 0.5 </td><td> 1 × 10 9 </td></tr><tr><td> P.プチダ KT2440 </td><td> KBMアンピシリン（100 ug / mlと） </td><td> 0.5 </td><td> 1 × 10 8 </td></tr><tr><td> A.ツメファシエンス GV2260 </td><td> LBリファンピシン（100 ug / mlと） </td><td> 0.5 </td><td> 5 × 10 8 </td></tr><tr><td> pstファイル DC3000 </td><td> KBMリファンピシン（100 ug / mlと） </td><td> 0.02 </td><td> 2 × 10 7 </td></tr><tr><td> pstファイルDC3000ΔhopQ1 - 1 </td><td> KBMリファンピシン（100 ug / mlと） </td><td> 0.1の100倍希釈</td><td> 1 × 10 6 </td></tr><tr><td> P.シリン PV。タバコ11528R </td><td> KBMリファンピシン（100 ug / mlと） </td><td> 0.1の100倍希釈</td><td> 1 × 10 6 </td></tr></tbody></table> 表2：培養条件およびアッセイで使用されている接種レベル。 ODと対応するCFUレベルは、分光光度計の異なるブランド間で異なる場合があります。

<ol>
	<li>Abramovitch, R. B., Anderson, J. C., Martin, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+elicitation+and+evasion+of+plant+innate+immunity.">Bacterial elicitation and evasion of plant innate immunity.</a> Nat Rev Mol Cell Biol. 7 (8), 601-60 (2006).</li><li>Jones, J. D., Dangl, J. 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+plant+immune+system.">The plant immune system.</a> Nature. 444 (7117), 323-323 (2006).</li><li>Zipfel, C., Robatzek, S., Navarro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+disease+resistance+in+Arabidopsis+through+flagellin+perception.">Bacterial disease resistance in Arabidopsis through flagellin perception.</a> Nature. 428 (6984), 764-764 (2004).</li><li>Oh, H. S., Collmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+resistance+against+bacteria+in+Nicotiana+benthamiana+leaves+is+accompanied+by+reduced+vascular+staining+and+suppressed+by+multiple+Pseudomonas+syringae+type+III+secretion+system+effector+proteins.">Basal resistance against bacteria in Nicotiana benthamiana leaves is accompanied by reduced vascular staining and suppressed by multiple Pseudomonas syringae type III secretion system effector proteins.</a> Plant J. 44 (2), 348-348 (2005).</li><li>Goodin, M. M., Zaitlin, D., Naidu, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotiana+benthamiana:+its+history+and+future+as+a+model+for+plant-pathogen+interactions.">Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions.</a> Mol Plant Microbe Interact. 21 (8), 1015-1015 (2008).</li><li>Wei, C. F., Kvitko, B. H., Shimizu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Pseudomonas+syringae+pv.+tomato+DC3000+mutant+lacking+the+type+III+effector+HopQ1-1+is+able+to+cause+disease+in+the+model+plant+Nicotiana+benthamiana.">A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana.</a> Plant J. 51 (1), 32-32 (2007).</li></ol>

細胞死ベースのアッセイは、PTIにおける遺伝子の関与を決定するために使用することができます。例えば、目的遺伝子の機能喪失型変異体は、このアッセイを用いてテストすることができます。インデューサーと、表1に与えられた課題の接種の4の組み合わせのうち、これが唯一の組み合わせは、ご使用のプラントバックグラウンドでの表現型をもたらすかもしれないことが可能です。我々はFLS2ために沈黙の植物は、インデューサーと挑戦接種のPF / DCとPf/Q1-1組み合わせでPTIの内訳（表1）を示すことを観察した。 それは、アッセイのための環境条件は、パート3.1で説明したものにできる限り近いことが重要です。低湿度では、スコアの分析が困難、あまりにも速く進行する細胞死を引き起こす。我々のプロトコルで記述された条件は、ラボでは動作しない場合は、インデューサーまたはチャレンジ接種のレベルを変更してみてください。我々は短い時間のギャップがアッセイはコントロール植物に早すぎると分解を引き起こしたことを発見したが、変更可能な別のパラメータには、誘導と課題間の時間差です。 我々は、少なくとも4〜5植物が実験でテストされると正の表現型は少なくとも3-4の独立した実験で繰り返されることをお勧めします。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>MES</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP300-100</td>
<td>Prepare as a1M stock, adjust pH and filter sterilize. Store at room temperature. </td>
</tr>
<tr>
<td>Life Science UV/Vis Spectrophotometer</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>DU 730</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

assay for pathogen-associated molecular pattern (pamp)-triggered immunity (pti) in plants

自分の環境で潜在的な病原体を認識するために、植物はそれらの形質膜上に存在するパターン認識受容体（PRRS）を使用してください。 PRRSは、病原体関連分子パターン（PAMPs）と呼ばれる保存された微生物の機能を認識し、この検出には効果的に非病原体の1,2により、植物組織の植民地化を防ぐPAMPトリガ免疫（PTI）、につながる。 PTIで最もよく研究システムは、FLS2依存性経路3です。 FLS2は、細菌のフラジェリンのコンポーネントであるPAMP flg22を認識します。 成功した病原体は病原性因子またはPTIを抑制し、病原体が病気1を引き起こすことができることができるエフェクターを持っている。順番にいくつかの植物は、エフェクターやエフェクタートリガ免疫（ETI）2につながる彼らの活動を、検出抵抗性遺伝子を持っている。 私たちは、ああ、Collmer 4から変更されたPTIのための細胞死ベースのアッセイを説明します。アッセイは、Nで規格化されました植物-病原体相互作用5の研究のためのモデル系としてますます使用されているbenthamiana、。 PTIは、葉への非病原性菌株の浸透によって誘導される。七時間後、どちらかの病気を引き起こすか、ETIを活性化する菌株は、オリジナルの浸透ゾーンをオーバーラップ領域に浸透しています。第一浸潤により誘導されたPTIは、第二の課題の浸潤に起因する細胞死の外観を遅らせたり、防止することができます。逆に、接種のオーバーラップ領域における細胞死の外観は、PTIの内訳を示している。 PTIとチャレンジ接種の誘導の4種類の組み合わせは、（表1）標準化した。アッセイは非サイレンシングNでテストされていますPTIを開発する能力が損なわれると予測されたFLS2のために沈黙の制御と植物を務めたbenthamiana植物 。

Watch this Scientific Journal Video about Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants at JoVE.com

Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

タバコbenthamiana植物のPTIのための細胞死ベースのアッセイが記載されている。

病原体関連分子パターン（PAMP）トリガ型植物におけるイミュニティ（PTI）のアッセイ

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize ...

assay-for-pathogen-associated-molecular-pattern-pamp-triggered

Research

JoVE Journal

Immunology and Infection

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

27.0K ビュー.  Boyce Thompson Institute for Plant Research. タバコbenthamiana植物のPTIのための細胞死ベースのアッセイが記載されている。

ビデオ: Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

の選択熱帯熱マラリア原虫</em人間の脳内皮細胞へのCytoadhesion用>寄生虫

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

免疫・感染学

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

使用肺炎球菌植民に対するプロバイオティクスの影響を調査<em&gt;体外</em&gt;接着アッセイ

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

せん断応力のヒト肝類洞内皮条件のことを白血球の募集を研究するフロー接着アッセイ

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

高収率の精製<em&gt;熱帯熱マラリア原虫</emオプソニン抗体アッセイにおける使用のための&gt;メロゾイト

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

治療薬にリンパ腫細胞の感度での役割を決定するために、好中球の単離と解析

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

蛍光ベースのノイラミニダーゼ阻害アッセイ抗ウイルスのノイラミニダーゼ阻害剤クラスにインフルエンザウイルスの感受性を評価します

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

ヘリコバクター ・ ピロリ感染と胃の病理のマウス ・ モデル

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

ウイルス感染とキイロショウジョウバエのホスト ウイルスの相互作用の解析

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

二本鎖 RNA と否定的な感覚の RNA のウイルス感染におけるパターン認識受容体の共焦点レーザー顕微鏡

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;