This work was supported by the Deutsche Forschungsgemeinschaft by grant Z within Sonderforschungsbereich 944 &#8216;Physiology and Dynamics of Cellular Microcompartments&#8217; and HE1964/18 within priority program 1580.

10nmの金ナノ粒子（ゴールドNPS）15の1の合成<ol><li>溶液Aを準備します160ミリリットルミリQ、または再蒸留水に2ミリリットル1％水溶液塩化金を追加します。 </li><li>溶液Bを準備します32ミリリットルミリQ、または再蒸留水に8ミリリットル、1％のトリクエン酸ナトリウム×2、H 2 Oおよび160μlの1％タンニン酸を追加します。 </li><li> 60℃に溶液A及びBをウォームアップし、攪拌しながら、それらを混合する。すぐに濃い青色を観察します。約15分後に赤色を観察します。その後、95℃まで加熱し5分間保持し、RTに溶液を冷却する。 </li><li>カーボンコーティングされたグリッド上にNP懸濁液のドロップを追加し、空気中で乾燥させる。透過型電子顕微鏡によってNPの大きさや形態を確認してください。 </li></ol>ローダミンN-ヒドロキシエステル（NHS）とBSAとラベリングゴールドNPの2コーティング16 <ol><li> 、1.5mlのエッペンドルフチュー​​ブに900μlの30分間15000×gで遠心機を金NPSを追加します。オペアンプtionally月一度に複数のチューブを準備します。 </li><li> 、900μlのペレットに滅菌ミリQ水を再懸濁し、100μlの2mg / mlのBSA / PBSを追加し、上清を捨て、30分間、800rpmでボルテックスに混ぜる。 </li><li>過剰のBSAを除去するために、60分間15000×gで準備を遠心する。 </li><li>上清を捨て、そして125μlのPBS中の金-BSA NPSを再サスペンド、1M重炭酸塩の12.5μlを添加する。 </li><li>使用直前に、金-BSA NPの懸濁液1ミリリットルするローダミンNHS / DMSO溶液30μlを添加、ローダミンNHS / DMSOの10 mg / mlの溶液を調製。光への暴露を避け、800rpmで混合中に室温で2時間（またはO / N）のための反応をインキュベートします。 </li><li> 48時間 - 36の期間にわたって5バッファ変更で4ºCでPBSに対して透析41612-BSA-ローダミンNPSを精製する。 </li><li> NPSを安定させるために、各1ミリリットル金-BSA-ローダミンのNPに200 mg / mlのBSA / PBSの10μlを添加する。 15で、無料またはBSA-ローダミンリリース遠心分離機を削除するには、60分間、000×gで。光への暴露を避け、4℃でOD 520、およびストアを測定する、2 mg / mlのBSA / PBSにペレットを再懸濁する。 </li><li> 、ミリQまたは二重蒸留水でNPを10倍に希釈炭素被覆グリッド上にドロップを追加し、空気中で乾燥させる。透過型電子顕微鏡（TEM）によるNPのサイズ及び形態を確認する。 NOTE：NPの調製のために使用されるすべての材料は、滅菌した操作は、細胞培養フード内または火炎横ベンチを行った。 </li></ol>ヒーラ細胞の3文化<ol><li>永久LAMP1-GFPを発現するHeLa細胞を、10％ウシ胎児血清（FCS）を含むDMEM中で培養し、5％CO 2を含む雰囲気中で37℃で増殖させている。 </li><li> 8ウェルチャンバースライド（Ibidi）においても50,000の密度で細胞を播種し、O / Nインキュベートする。 </li></ol>細菌の4。文化<ol><li>使用サルモネラ·エンテリカ血清型TyphimuriumのNCTC 12023ウィルd軸型（WT）株。比較のために、SIFのキーエフェクターSIFAを欠いSPI2-T3SSとΔSIFAにおける欠損変異株のΔssaVを使用しています。強化されたGFPの構成的発現のためのプラスミドpFPV25.1を保有する菌株を使用してください。 注：細菌株は、50μg/ mlのカルベニシリンおよび/ ​​または50μg/ mlのカナマイシン（ΔssaV、ΔSIFAを追加してルリア-ベルターニの50μg/ mlのカルベニシリン（WT）を添加した培養液（LB）とLB中で日常的に培養され、 ）プラスミドを維持するために必要である。 </li><li>適切な抗生物質で3ミリリットルのLB中の細菌の単一コロニーを接種し、新鮮なLBで1：31に希釈し、3.5時間のために成長を続ける、その後、通気のための条件をとうしながら37ºCでO / Nを育てる。この時点で、培養物を後期対数期に達し、細菌は非常に侵襲的である。 「ローラードラムは「通気試験管培養物をインキュベートするのが便利である。 </li></ol>によるHeLa細胞の感染5。サルモネラとゴールド-BSA-ローダミンNPは、チェース·ラベリングパルス（スキームについては図1を参照） <ol><li>継代培養した細菌のOD 600を測定し、OD 600に希釈= 0.2mlの1 PBS（〜3×10 8 cfu / mlで）、多数のを達成するために、8ウェルチャンバースライド中のHeLa細胞への細菌の適切な量を追加100の感染（MOI）。 </li><li> （この時点を0時間感染後、0時間のπとした）の非内在化細菌を除去するためにPBSで3回洗浄し、細胞インキュベーター中で30分間インキュベートする。 100μg/ mlのゲンタマイシンを含有する新鮮な培養液の300μl添加し、1時間維持する。その後、インキュベーション時間の残りのための10μg/ mlのゲンタマイシンを含有する新鮮な培地で培地を交換してください。 </li><li> 1時間、100μg/ mlのゲンタマイシンを含有する培地とのインキュベーション後、培地を同時（30mMのHEPES、pH7.4で、FCS、L-グルタミン、フェノールレッド、および炭酸水素ナトリウムなしイーグルMEM）培地を撮像して置換されている10μg/ mlのゲンタマイシンをntaining。金BSA-ローダミンNPはOD 520 = 0.1の最終濃度を得るために、HeLa細胞に添加される。 NOTE：金BSA-ローダミンNPはまた、感染の前に、または種々の時点のπで細胞に添加してもよい</li><li> 1時間のインキュベーション後、培地を除去し、PBSで3回洗浄し、インキュベーション時間の残りの10％のFCSおよび10μg/ mlのゲンタマイシンを含有する新鮮な画像形成媒体の300μlを添加する。 注意：インキュベーションの時間は、使用のNPおよび細胞株の濃度に依存して変化し得る。 RAW264.7マクロファージに関しては、十分な内在許可OD 520 = 0.05の濃度のNPとその30分のインキュベーションを観察した。 </li></ol> 6.イメージング<ol><li>共焦点レーザー走査（CLSM）またはスピニングディスク共焦点イメージングシステムの異なる時点での高分解能イメージングのための加湿環境チャンバーと（SD）顕微鏡を使用する。 </li><li>温度Cに切り替えontrolシステム、それが安定するまで待ちます。そのような倍率、スキャン速度、分解能、ZスタックなどGFPのために適切な励起/発光の設定を使用し、金-BSA-ローダミンNPのような画像の設定を最適化します。このプロトコルのために、512×512ピクセルの400 Hzのスキャン速度、分解能および0.25μmのZ-ステップサイズを使用。それぞれ、Arレーザ（488 nm）をHeNeレーザー（543 nm）を用いて、GFPと金-BSA-ローダミンエキサイト。セルの形状を観察するための明視野（BF）のチャンネルを含める。他の顕微鏡システムおよび感染条件については、設定に応じて調整されなければならない。 NOTE：BFチャネル信号の光源がフォトマルチプライヤ（PMT）検出器で検出されたのと同じArレーザを使用する。 </li><li>指示された時点のPIでは、顕微鏡ステージと記録画像に感染した細胞を含む8ウェルチャンバースライドをマウントします。 </li></ol>画像の7.分析<ol><li>顕微鏡画像解析ソフトウェアを使用して（参照<stronG&gt;材料および装置の表）は、細胞内サルモネラによるエンド-リソソームシステムの再構築を分析する。 「開く」をクリックし、ファイルを選択してデータを開きます。 注：あるいは、このようなImageJのかFIJIなどのオープンソース·ソフトウェア·パッケージは、定量的画像解析に使用することができる。 </li><li>アイコンにビューを突破クリックのオブジェクトツールバーで<img alt="アイコン" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon1.jpg" />新しい表面アイテムを追加します。上をクリック<img alt="アイコン" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> （次）。 </li><li>関心領域（ROI）を選択し、セグメントROIを分析する。表示エリアには、画像に重ね矩形ボーダー部にROIを表している。対応するX軸、Y軸、Z-フィールドに値を入力するか、直接ROIのサイズと位置を変更するために、プレビューの長方形の矢印をクリックしてください。クリック<img alt="アイコン" fo:content-width=".1in" src<html> = &quot;/ファイル/ ftp_upload / 52058 / 52058icon2.jpg&quot; /&gt;（次へ）。 </li><li>ソースチャネルとして、セレクトチャンネル3（ゴールド-BSA-ローダミンNPのための信号）。結果の面積の滑らかさを設定するには、円滑なオプションをチェックします。手動で値を定義するか、自動的に生成された値を受け入れます。しきい値の、絶対強度オプションを選択します。 </li><li>閾値調整のために、手動オプションを選択して、値を設定します。視域では、表面閾値プレビューは灰色で表示されている。 </li><li>タブ分類表面に得られた表面は、様々な基準によってフィルタリングすることができます。デフォルトのフィルタは「&gt; 10ボクセルの数」、および他のフィルタは、「追加」をクリックして含めることができている。フィルタリングが必要でない場合は、[削除]ボタンをクリックして、すべてのフィルタを削除します。クリック<img alt="アイコン" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> （次）。 </li><li>サーフェス作成を完了するには、上をクリック/files/ftp_upload/52058/52058icon3.jpg &quot;/&gt;（仕上げ）を。オブジェクトのリストで、現在、アイテム·ボリュームと新しい作成表面について、未チェックボックスが表示領域に表示されます。 </li><li>統計情報をエクスポートします。さて、突破ビューで被験者の色は、その面積に応じて紫から赤に変わる。そして、「プロット番号エリア」テーブルには、統計情報（ 例えば、ID番号とオブジェクトの面積）を示している。クリックでExcelファイルに面1のすべての統計情報をエクスポート<img alt="アイコン" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon4.jpg" /> （保存）。 </li></ol>

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No conflicts of interest declared.

哺乳動物細胞のエンドリソソーム系は、栄養吸収、ホルモン媒介性シグナル伝達、免疫監視、および抗原提示17を含む重要な生理的過程を制御する。これまで、種々のマーカーは、エンドサイトーシス経路および追跡調査を標識するために使用されてきた。例えば、リソトラッカープローブは、選択的に低い内部pHの細胞区画に蓄積し、効果的にナノモル濃度18に住んで細胞を...

金属などのNP、量子ドット、ポリマーのNP、シリカNPは、カーボンドットを含む、蛍光ナノ粒子（NPは）、過去数十年の間、1,2-かなりの注目を集めている。従来の有機染料に比べて、蛍光性NPは、このような強力な信号強度、光退色に対する抵抗性及び生体適合性の高い3,4のような望ましい化学的、光学的および機械的特性を示す。これらの利点は、それらの細胞内のセンシングと生細胞イメージングのための選択の方法にする。さらに、電子密度の高いNPの様々な生細胞の組み合わせは、光学顕微鏡（LM）とEM 5と超微細構造レベルでのより高い分解能でトラッキングすることができ、相関顕微鏡分析のためのそれらの使用を容易にする、電子顕微鏡（EM）から見える。例えば、金NPは、効率的に感受性の診断のための生細胞におけるバイオセンサーとして、ならびに免疫標識6の分野で使用される長い時間であった。最近のStudiesは、異なるサイズおよび形状を有する金NPが容易に大きな電位が細胞内小胞輸送の追跡およびエンドリソソーム系標識7,8を申請しているため、細胞株の大様々な取り込みおよび日常エンドソーム経路を介して輸送され得ることを示す。 そのようなサルモネラ·エンテリカ 、 赤痢菌およびリステリアなどの微生物病原体は、非食宿主細胞9に侵入するために異なるメカニズムを開発しました。内在化された後に、病原体のいずれかのサイトゾルに局在または膜結合区画に隔離は、それらのホスト環境で広範囲に相互作用して、自分の生存10を優先するように、これらを調節する。例えば、 サルモネラ·エンテリカは、細胞内ファゴソーム区画内に存在し、複製し、感染11上にサルモネラ含有液胞（SCV）と呼ばれる。成熟SCVエンドサイトーシス経路で連続的相互作用を受けてゴルジ装置に向かうトラフィック、およびサルモネラ誘発フィラメント（SIF）などの大規模な管状構造の形成を誘導するが、ネキシン細管ソート、 サルモネラ、分泌キャリア膜タンパク質3（SCAMP3）細管など誘発。12-14。これらの細菌性病原体が宿主細胞の経路を操作する方法勉強する感染症を理解するために不可欠です。 ここで、金BSA-ローダミンNPは、宿主細胞のエンドリソソーム系を標識するために流体トレーサーとして使用し、ヒトの胃腸病原体サルモネラ·エンテリカ血清型ネズミチフス菌（ サルモネラ ）と病原体の相互作用を研究するためのモデル菌として使用したエンドサイトーシス経路をホストします。 WT サルモネラまたは突然変異株を感染させた非感染細胞と細胞の細胞内の金BSA-ローダミンNPを、共焦点レーザー走査顕微鏡（CLSM）で画像化した。その後IMARISソフトウェアはサルモネラ感染がエンドソーム/リソソームの極端な再配列を誘導したことを示す、NPの分布を定量した。この方法の説明に続いて、類似の実験は、内在化NPの長期の運命を追跡し、真核細胞のエンドサイトーシス経路上の様々な外因性物質または内因性因子の影響を調査するために設計することができる。

<table border="0" cellpadding="0" cellspacing="0" width="684">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:193px;">Name of the Material/Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:149px;">Catalog Number</td>
			<td style="width:176px;">Comments/ Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gold chloride</td>
			<td>Sigma-Aldrich</td>
			<td>520918</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tannic acid</td>
			<td>Sigma-Aldrich</td>
			<td>403040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tri-sodium citrate</td>
			<td>Sigma</td>
			<td>C8532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NHS-Rhodamine</td>
			<td>Pierce</td>
			<td>46406</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMSO&#160;</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamicin</td>
			<td>Applichem</td>
			<td>A1492</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamcyin</td>
			<td>Roth</td>
			<td>T832</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carbenicillin</td>
			<td>Roth</td>
			<td>6344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">8-well chamber slides</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td>tissue culture treated, sterile</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaris Software</td>
			<td>Bitplane</td>
			<td>version 7.6</td>
			<td>various configurations available</td>
		</tr>
	</tbody>
</table>

application of fluorescent nanoparticles to study remodeling of the endo-lysosomal system by intracellular bacteria

望ましい化学的、光学的および機械的特性を有する蛍光ナノ粒子（NPが）、細胞内小器官を標識するための有望なツールである。ここでは、真核細胞のエンド-リソソームシステムにラベルを付け、細胞内病原体サルモネラ·エンテリカによりホスト細胞経路の操作を監視するために金-BSA-ローダミンNPSを使用する方法を紹介します。 NPは容易に後期エンドソーム/リソソームにHeLa細胞によって内在して局在していた。 サルモネラ感染症はSalmonella-誘起膜構造における小胞およびNPの蓄積の再配置を誘導した。我々は、共焦点顕微鏡画像の定量分析のためのIMARISソフトウェアパッケージを展開。非感染細胞内のオブジェクトの数とそれらのサイズ分布は、WT サルモネラによってエンドリソソーム系の非常にリモデリングを示す、 サルモネラ感染細胞におけるものとは異なっていた。

金NPは、クエン酸、タンニン酸による塩化金酸の還元を介して、十分に確立された方法により作製した。 図2Aに示すように、合成された金NPは、約10nmのサイズを有する形状に準球形であった。 BSA-コーティングおよびローダミン標識は、それらの形態や大きさ（ 図2B）に影響しませんでした。 これは、金NPは容易に種々の哺乳動物細胞により取り?...

Watch this Scientific Journal Video about Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria at JoVE.com

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

この記事では、合成とナノ粒子（NPS）の蛍光標識するための方法を説明します。 NPは、真核細胞のエンドリソソーム系を標識するために、パルスチェイス実験に適用した。細胞内病原体サルモネラ·エンテリカの活動によって、エンド-リソソームシステムの操作は、ライブセルイメージングが続き、定量化した。

細胞内細菌によって遠藤-リソソームシステムの再構築を検討するために、蛍光ナノ粒子の応用

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

application-fluorescent-nanoparticles-to-study-remodeling-endo

Research

JoVE Journal

Immunology and Infection

9.9K ビュー.  Universität Osnabrück. この記事では、合成とナノ粒子（NPS）の蛍光標識するための方法を説明します。 NPは、真核細胞のエンドリソソーム系を標識するために、パルスチェイス実験に適用した。細胞内病原体サルモネラ·エンテリカの活動によって、エンド-リソソームシステムの操作は、ライブセルイメージングが続き、定量化した。 

ビデオ: Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

オリゴヌクレオチド結合蛍光マイクロスフェアを使用して複雑な臨床及び環境試料中の細菌の多重検出

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

免疫・感染学

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

ホット水 - フェノール抽出によるグラム陰性菌からの精製とリポ多糖の可視化

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

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 ...

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

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

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

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

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

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

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

誘導発現系を適用すると、細胞内シグナル伝達と細菌の病原性因子の干渉を研究するために、

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

「Acufection」と肌の研究への応用により、経済的なDNAデリバリーシステムの開発

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

細胞内の標的阻害剤の有効性及び細胞毒性スクリーニングのためのシステム<em&gt;結核菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

生体外でマウスのナチュラル キラー細胞の大量生産のため新規無料の送り装置システム

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic purposes. However, the visualization and quantification of multiple antigens in a single tissue section using conventional chromogenic IHC is challenging. Multiplexed imaging is especially relevant in lymphoma research and diagnostics, where markers have to be interpreted in the context of a complex tumor microenvironment. Here we describe a protocol for multiplexed fluorescent IHC staining to enable the quantitative assessment of multiple targets in specific cell types of interest in lymphoma.The method covers aspects of antibody validation, antibody optimization, the multiplex optimization with markers of lymphoma subtypes, the staining of tissue microarray (TMA) slides, and the scanning of the slides, followed by data analysis, with specific reference to lymphoma. Using this method, scores for both the mean intensity of a marker of interest and the percentage positivity are generated to facilitate further quantitative analysis. Multiplexing minimizes sample utilization and provides spatial information for each marker of interest.

Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections.

多重蛍光免疫組織化学染色、イメージング、およびリンパ腫の組織学的試料中の分析

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic ...

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

嫌気性増殖細菌によるマウス膣コロニー形成モデル

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...