This work was supported by NIGMS, National Institutes of Health (GM094623). Significant portions of this work were performed in the Environmental Molecular Sciences Laboratory, a DOE/BER national scientific user facility located at the Pacific Northwest National Laboratory (PNNL). PNNL is operated for the DOE by Battelle under Contract DE-AC05-76RLO1830.

1.事前に準備します<ol><li> 37℃の温かい滅菌リン酸緩衝生理食塩水（PBS）。 </li><li> 10％ウシ胎児血清（FBS）を補充したイーグル培地（DMEM）および最小必須培地（MEM）の温かいダルベッコ改変、100 IU / mlペニシリン、および100μg/ mlストレプトマイシン、37℃である。注：これらはそれぞれ、RAWおよびHeLa細胞のために正常な成長メディア（NGM）を表す。 </li></ol>細胞の調製<ol><li> NGMで百分の70から90までの集密度にRAW 264.7細胞を増殖させる。 <ol><li>加湿95％空気/ 5％CO 2雰囲気中、37℃で細胞を維持する。 </li></ol></li><li> NGMで70から90パーセントの集密度にHeLa細胞を成長させる。 <ol><li>加湿95％空気/ 5％CO 2雰囲気中、37℃で細胞を維持する。 </li></ol></li><li>コレクションの前に、滅菌PBSで一度細胞単層を洗う。 </li><li>無菌コニカルチューブにあらかじめコンフルエント細胞を収集する。 <ol><li>静かにRAW細胞をこすりPBSでラバーポリスで、繰り返しピペッティングで細胞集合体を分散させる。 </li><li>目視検査は、培養表面から解離を示すまで、0.25％トリプシン溶液を用いてHeLa細胞を切り離します。例えば、T-75フラスコのために2〜3ミリリットルを使用しています。トリプシン溶液は、全体の成長表面を覆っていることを確認するためにそれに応じて音量を調整します。 <ol><li> NGMは、10％FBSを含有すると解離反応をクエンチした。 </li><li>セル集計を分散させるために繰り返しピペット操作で、トリプシンNGMの少なくとも2倍のボリュームを使用してください。 </li></ol></li></ol></li><li>軽く4分間900×gでの遠心分離によって細胞をペレット。 </li><li>滅菌PBSを用いてトリプシン/停止溶液と同じ体積で再懸濁する。 </li><li>血球計数器またはパーティクルカウンターを用いて細胞数を計測する。 </li><li>軽く4分間900×gでの遠心分離によって細胞をペレット。 </li><li> 5.5×10 6×10 6〜6.0個/ mlのためのPBSの十分な音量で再懸濁します。注：例えば、T-75フラスコapproximを生じる。ately 7.5×10 6細胞29。 </li><li>エレクトロポレーションまで氷上に細胞懸濁液を保管してください。 </li></ol>エレクトロポレーションのための3.準備<ol><li>氷上で事前チルエレクトロポレーションキュベット（0.4cmのギャップ）。 </li><li>エレクトロポレーション装置の電源を入れ、0.3 kVの電圧を設定する。 注：容量と抵抗が私たちのエレクトロ（メーカー10μFと600Ωでのセット）で調整可能な設定はありませんでした。 </li><li> NGMで回復プレートを記入し、37℃の加湿95％空気/ 5％CO 2雰囲気中で平衡化</li></ol> 4.エレクトロポレーション<ol><li> （50μgの/ ml）を予め冷却したキュベット中の細胞懸濁液の400μlのを置き、キュベットに選択されたタンパク質20μgのを追加します。 </li><li>フリックキュベットを穏やかに約10回の細胞を損傷させることなく混合する。注：キュベットも完全に混合するために数回反転させてもよいが、ピペッティングしないとダウンしているかボルテックス細胞の損傷を防ぐために。 </li><lエレクトロに電気アーク放電を回避するためにペーパータオルまたは他のワイパーとのキュベットのI&gt;ドライ外に。 </li><li> 1.5から1.7ミリ秒0.3 kVのでエレクトロポサンプル。注：これは、この研究のために典型的だった。 </li><li>すぐにエレクトロポレーションフリックキュベット後に優​​しく〜10回を徹底的に混合する。 </li></ol>細胞の5。メッキ<ol><li>回収プレートで配置する直前まで、氷上でエレクトロポレーションした細胞を持つストアキュベット。 </li><li>最下流の分析のために、余分なエフェクタータンパク質を除去するために予め温めたNGMで細胞を1倍洗う。 <ol><li>分析のための細胞を除去し、NGM 3-5 mlの一時停止。 </li><li> 4分間900×gでの遠心分離によって細胞をペレット。 </li><li>所望のプレートサイズのNGMの適切な容積に再懸濁（35mmディッシュ用など 2ml）中。 </li></ol></li><li>下流の分析のための細胞の適切な量を削除します。 <ol><li>例1：顕微鏡分析用ガラスボトムディッシュにプレート。 </li><li>例2：プレートINTそのような親和性精製などのタンパク質分析oを細胞培養プラスチック。 </li></ol></li><li>細胞は、少なくとも4時間、37℃で加湿した95％空気/ 5％CO 2雰囲気中で平衡化プレートに回復させる。 </li></ol> 6.顕微鏡分析 6.1）固定/免疫蛍光染色<ol><li>洗浄細胞を4時間の回復期間の後、滅菌PBSで1倍。 </li><li>室温で2分間、100％メタノールで細胞を固定する。完全に細胞（35mmディッシュ用など 2ミリリットル）をカバーするのに十分なメタノールを使用してください。 </li><li>滅菌PBSで洗浄3X。 </li><li> 15分間、PBS中0.4％トリトンX-100で細胞を透過性。例えば、直径35mmの板に1ミリリットルを使用しています。 <ol><li>エピトープおよび標的タンパク質の位置に応じてトリトンX-100の透過及び強度の長さを調整する。注意：最良の結果はしかし、上記の条件は、ほとんどのCで十分であるべきであり、経験的に各ターゲットのために決定する必要がありますytosolicターゲット。 </li></ol></li><li> RTで1時間、PBS中の5％ウシ血清アルブミン（BSA）でブロックする。例えば、直径35mmの板に1ミリリットルを使用しています。 </li><li> PBSで洗浄3X。 </li><li> 4℃で一晩、抗体結合溶液中で一次抗体（PBS中0.1％トリトンX-100および1％BSA）をインキュベート&#160;穏やかに揺らしながら°C。例えば、直径35mmの板に0.5ミリリットルを使用しています。 <ol><li>抗体希釈用の製造元の推奨に従ってください。注：200希釈PKN1抗体は1で使用しながら1,000希釈たとえば、ストレプトアビジン結合ペプチドタグ（SBPタグ）抗体は1で使用した。 </li></ol></li><li> PBSで洗浄4X。 </li><li>光から保護し、室温で1時間、抗体結合溶液中で適切な蛍光結合二次抗体と共にインキュベートする。 <ol><li>例えば、室温で1時間、アレクサ488またはアレクサ647を使用する。 <ol><li>アリのためのメーカーの推奨事項に従ってくださいibody希釈。 （ 例えば 、約1：この研究のために1,000）。 </li></ol></li><li>例えば、必要に応じて他の汚れを追加し、5μM小麦胚芽agglutinnin（WGA）を室温で1時間、製造業者の推奨に従ってアレクサ647またはDAPIにコンジュゲート。 </li></ol></li><li>画像への準備が整うまで、光から保護し、PBSおよび4℃で店を洗浄5回、。 </li></ol> 6.2）共焦点顕微鏡と画像解析<ol><li> 63×油浸対物レンズを用いた倒立共焦点顕微鏡での画像サンプル。 <ol><li> 492から542 nmの間の帯域幅の放射を有するアルゴンレーザーの488nmのラインを使用して画像の緑色チャネル、。 </li><li> 640から718 nmの間の帯域幅の放射を、633nmのダイオードレーザーを使用して、画像の赤チャンネル。 </li><li> 407から453 nmの間の帯域幅の放射を、405nmのダイオードレーザーを用いて画像青チャンネル。 </li><li> Z-スタックは細胞容積の全体をカバーする複数のチャンネルを確認してください。 </li></ol></li><li>適切な画像処理ソフトウェアで処理·イメージ。 </li></ol> 7.アフィニティ精製<ol><li> 4時間の回復期間の後、4℃PBSで2回エレクトロポレーションした細胞を洗浄。 </li><li>氷上で溶解緩衝液（PBS中のプロテアーゼ阻害剤カクテルおよびホスファターゼ阻害剤1％トリトンX-100）の〜1.0ミリリットルと溶解する。製造業者の指示に従って、阻害剤を使用してください。注：たとえば、両方の本研究で用いたホスファターゼおよびプロテアーゼ阻害剤は、100倍で供給したが、他の製剤は、同様に良好に動作するはずです。 </li><li>速やかにラバーポリスで細胞をこすりし、円錐形のチューブに集める。 </li><li>激しいボルテックスおよび超音波処理によって溶解する。注：他の溶解方法は、同様に同様に動作する可能性がありますが、効率は経験的に分析要件の適合性として決定する必要があります。 <ol><li>断続的にボルテックスしながら超音波処理3×30秒。 </li></ol></li><li>収集するために4℃で10分間万×gで遠心分離し細胞破片と不溶性の凝集体。上清を保存します。 </li><li>転倒回転で4℃で一晩ストレプトアビジンアガロース樹脂懸濁液50μlでエレクトロ細胞溶解物の等容量（〜1.0ミリリットル）を組み合わせる。注：この樹脂は、そのストレプトアビジン結合ペプチドの親和性タグを経由して電気穿孔したタンパク質（および関連複合体）をキャプチャします。 </li><li> 2分間2500×gで遠心分離し、上清を捨てる。 </li><li> 40床体積（〜1ミリリットル）PBSで2回洗浄します。 </li><li> 30μlの4X LDSローディングバッファー（141 mMのトリス塩基、2％LDS、10％グリセロール、0.51 mMのEDTA、0.22 mMのSERVAブルーG、赤0.175 mMのフェノール、pH8.5）中、20μLのdiH 2 O、および1μlのを追加します。いくつかのボリュームを可能にする0.5 Mトリス（2-カルボキシエチル）ホスフィン（TCEP）は、ビーズ中に残留する。 </li><li> 10分間95℃で加熱し、氷上で冷却する。 </li><li> 5分間、4℃で&gt;万XGスピン。 </li><li>上清を収集します。 </li><li>適切な抗体を用いたウエスタンブロットを実行注：彼再度、抗PKN1一次抗体が使用される。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+of+electroporation:+A+review.">Theory of electroporation: A review.</a> Bioelectrochemistry and Bioenergetics. 41, 135-160 (1996).</li><li>Weaver, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+theory.+Concepts+and+mechanisms.">Electroporation theory. Concepts and mechanisms.</a> Methods in Molecular Biology. 55, 3-28 (1995).</li><li>McAteer, J. A., Douglas, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monolayer+culture+techniques.">Monolayer culture techniques.</a> Methods in Enzymology. 58, 132-140 (1979).</li><li>Ho, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+GtgE,+a+novel+virulence+factor+encoded+on+the+Gifsy-2+bacteriophage+of+Salmonella+enterica+serovar+Typhimurium.">Identification of GtgE, a novel virulence factor encoded on the Gifsy-2 bacteriophage of Salmonella enterica serovar Typhimurium.</a> Journal of Bacteriology. 184, 5234-5239 (2002).</li><li>Niemann, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+novel+secreted+virulence+factors+from+Salmonella+enterica+serovar+Typhimurium+by+proteomic+analysis+of+culture+supernatants.">Discovery of novel secreted virulence factors from Salmonella enterica serovar Typhimurium by proteomic analysis of culture supernatants.</a> Infection and Immunity. 79, 33-43 (2011).</li><li>Spano, S., Liu, X., Galan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+targeting+of+Rab29+by+an+effector+protein+distinguishes+the+intracellular+compartments+of+human-adapted+and+broad-host+Salmonella.">Proteolytic targeting of Rab29 by an effector protein distinguishes the intracellular compartments of human-adapted and broad-host Salmonella.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 18418-18423 (2011).</li><li>Haraga, A., Miller, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Salmonella+type+III+secretion+effector+interacts+with+the+mammalian+serine/threonine+protein+kinase+PKN1.">A Salmonella type III secretion effector interacts with the mammalian serine/threonine protein kinase PKN1.</a> Cellular Microbiology. 8, 837-846 (2006).</li><li>Zinchuk, V., Grossenbacher-Zinchuk, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+colocalization+analysis+of+confocal+fluorescence+microscopy+images.">Quantitative colocalization analysis of confocal fluorescence microscopy images.</a> Current Protocols in Cell Biology / Editorial Board, Juan S. Bonifacino ... [et al]. 4 (Unit 4.19), (2011).</li><li>Kimple, M. E., Sondek, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+affinity+tags+for+protein+purification.">Overview of affinity tags for protein purification.</a> Current Protocols in Protein Science / Editorial Board, John E. Coligan ... [et al]. 9 (Unit 9.9), (2004).</li></ol>

著者らは、開示することは何もない。

病原性細菌から分泌されたエフェクターは、宿主細胞環境内で機能するように進化してきたので、それは、宿主内で、その場でそれらを研究するために有用である。宿主細胞への関心の特定のエフェクターの導入は、関連する病原体 - 宿主相互作用は、他の細菌タンパク質からの干渉なしに孤立して研究することができるようになります。目標は、それによってトランスフェクション?...

多くのグラム陰性細菌に直接、宿主細胞中に1-5（エフェクターと呼ばれる）病原性関連タンパク質を注入するために特殊な分泌系を使用する。宿主免疫の抑制、細胞骨格の変化、細胞内輸送およびシグナル伝達、転写変化の修正、およびホストプロテアソーム改変6-9：これらのエフェクターを含む広範囲の生物学的機能を有している。いくつかのエフェクターの機能は、しかし、ホスト·ターゲットおよび多くの他の生化学的作用（複数の）決定されていない、知られている。野生型および組換え細菌感染を比較する細胞内エフェクター病原性メカニズムを研究するための有効なアプローチであるが、それは、宿主細胞に個別のエフェクターを導入することが有利であることが多い。したがって、宿主細胞の文脈における細菌エフェクタータンパク質を導入し、特徴づけるための単純な方法が非常に望ましい。 実験的な分析のWiの簡素化単一エフェクターが重要な番目他のエフェクターは、対向または冗長機能を有していてもよいように。この単純化を達成するために、研究者は、以前にウイルス形質導入10、マイクロインジェクション11を含む多くの異なる方法によって細胞内に巨大分子を導入して、12,13のロードこすり、化学的に誘導されマイクロインジェクション14による細胞融合、独自のタンパク質「トランスフェクション」は、試薬15、リン酸カルシウム沈殿16、およびエレクトロポレーション17-20。導入された分子は、タンパク質、細胞不透過性色素、および細胞内ターゲット21,22のための抗体のDNA、RNA、およびRNAiの種を含む核酸の範囲である。いくつかの方法を導入することができる高分子の種類などの制限があり、特に、下流の分析は、高い細胞毒性、作用機序の損傷、低効力、または導入効率に制限されてもよい。トランスフェクション、ofte哺乳動物細胞中で細菌遺伝子を発現させるためのnに使用される方法は、また、マクロファージおよび初代細胞のようないくつかの関連する宿主細胞型は、トランスフェクションに向かって特に耐性であるという制約を受ける。これを超えて、それは、外来DNAの導入の際に生成細菌タンパク質のレベルを制御することは困難である。 多くの研究は、一般的な実験手法として、細菌および哺乳動物細胞の両方への核酸の電気穿孔法を確立した。しかし、生理的条件下で細胞内​​にタンパク質を送達するための最良の方法に進行中の研究がある。タンパク質のトランスフェクションに関する報告は有望であるが、高価な試薬及び最適化を必要とする。最小限のコストで細胞標的の多種多様な潜在的に有毒な細菌のエフェクターを導入する欲求は、生体内でこれらのタンパク質を研究するための方法として、エレクトロポレーションを検討するために私達を導いた。 タンパク質のエレクトロポレーション23-25 ​​が満たされるまた、電気トランスフェクションまたは電気注射26として知られているelectropermeabilizationを経由して生きている細胞内にタンパク質を導入するHOD、。この技術は、細胞膜に細孔を作成するために高強度の電気パルスを使用する。これらの可逆細孔は、通常、細胞内空間から除外されている巨大分子が細胞を入力することができます。外部電界が除去されると、膜は、細胞は細孔27,28を通過した分子を保持することができ、再シールすることができる。 標準的なエレクトロは一貫マウスマクロファージ様細胞およびヒト上皮細胞の両方に細菌のエフェクターを導入するために本研究で使用した。この方法は、細胞の生存率のかなりの減少と、迅速、効率的、かつ安価である。導入されたタンパク質は、免疫蛍光顕微鏡で可視化または機能的アッセイのために使用することができる。これは同様に、非毒性基準として緑色蛍光タンパク質（GFP）を使用して実証されている2 サルモネラエフェクタータンパク質、SspH1とGTGE。私たちは、真核宿主細胞における細菌の病原性タンパク質とその機能の研究のためのレパートリーに追加のツールと​​してタンパク質のエレクトロポレーションを提案する。

<table border="0" cellpadding="0" cellspacing="0" width="1132">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:532px;">Material or Equipment</td>
			<td style="width:177px;">Company</td>
			<td style="width:108px;">Catalog Number</td>
			<td style="width:315px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.25% Trypsin-EDTA Solution</td>
			<td>Cellgro</td>
			<td>25-050-Cl</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.4cm Gap-disposable electroporation cuvettes</td>
			<td>Bio-Rad</td>
			<td>165-2088</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100% Methanol</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Flammable, Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A4919</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Counting Apparatus - Hemocytometer or Coulter Counter</td>
			<td>Beckman Coulter</td>
			<td>Model Z1</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cell Culture Incubator</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Humidified 95% air/5% CO2 atmosphere at 37 &#176;C&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Culture Plastic</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Cell culture flasks/plates, pipets, tubes, rubber policeman</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modification of Eagle&#8217;s Medium (DMEM)</td>
			<td>Cellgro</td>
			<td>10-013</td>
			<td>Warm to 37 &#176;C&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Electroporator</td>
			<td>Bio-Rad</td>
			<td>E. coli Pulser</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Cellgro</td>
			<td>35-016-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescent confocal microscope&#160;</td>
			<td>Ziess</td>
			<td>Model&#160; LSM 710&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Bottom Dishes for Microscopy</td>
			<td>Wilco Wells</td>
			<td>HBSt-3522</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HALT Protease Inhibitor Cocktail</td>
			<td>Pierce</td>
			<td align="right">78430</td>
			<td>Corrosive, Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HeLa Cell Line</td>
			<td>ATCC</td>
			<td>ATCC CCL-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LDS 4X Loading Buffer</td>
			<td>Invitrogen</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimal Essential Medium (MEM)&#160;</td>
			<td>Cellgro</td>
			<td>10-010</td>
			<td>Warm to 37 &#176;C&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Other fluorescent stains (WGA, DAPI) in conjunction with anti-fade reagent</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin</td>
			<td>Cellgro</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RAW 264.7 Cell line</td>
			<td>ATCC</td>
			<td>TIB-71</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Primary Antibody Against Target of Interest</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Secondary Antibody Conjugated to Fluorophore</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Chill to 4 &#176;C&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile Phosphate Buffered Saline</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Warm to 37 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Agarose Resin Suspension&#160;</td>
			<td>Pierce</td>
			<td align="right">20353</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Table Top Centrifuge Capable of Accepting Conical Tubes (swinging bucket preferred)</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TCEP</td>
			<td>Sigma-Aldrich</td>
			<td align="right">646547</td>
			<td>Corrosive, Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8585</td>
			<td>Irritant, Toxic</td>
		</tr>
	</tbody>
</table>

electroporation of functional bacterial effectors into mammalian cells

生きた細胞の文脈におけるタンパク質相互作用の研究は、ローカリゼーション、ダイナミクス、相互作用パートナーに関する重要な情報を生成することができます。この情報は、宿主 - 病原体相互作用の文脈において特に価値がある。多くの病原体タンパク質は、細胞内環境内で宿主免疫系および生存の回避を可能にするような方法で、種々の宿主細胞内で機能する。タグ化または変異タンパク質、またはトランスフェクションまたは形質導入を介して病原体遺伝子の導入を発現する株によるインビボ感染これら病原体タンパク質の宿主細胞間相互作用を研究するために、いくつかのアプローチは、一般的に含め、使用されている。これらのアプローチの各々は、長所と短所がある。私たちは、直接細胞に外因性タンパク質を導入する手段を求めた。エレクトロポレーションは、一般的に細胞に核酸を導入するために使用されるが、生物物理学的根拠は全く同じであるが、それ以上のことはほとんどのタンパク質に適用されていない。標準的なエレクトロポレーターは、哺乳動物細胞への親和性タグ化細菌のエフェクターを導入するために使用した。ヒト上皮及びマウスマクロファージ細胞は、従来の方法により培養剥がし、目的の外来細菌性病原体タンパク質（ 例えば、ネズミチフス菌GTGE）と0.4cmのギャップエレクトロポレーションキュベットに入れた。電気穿孔法（0.3 kVの）および短い（4時間）の回復期間の後、細胞内タンパク質は、蛍光の親和性タグを介してタンパク質を標識し、共焦点顕微鏡によって空間的および時間的分布を調べることにより確認した。エレクトロポレーションしたタンパク質はまた、細胞内の機能的かつ正確な細胞内輸送およびタンパク質 - タンパク質相互作用が可能であることが示された。外因性タンパク質は、細胞の表面上に蓄積する傾向があったが、エレクトロポレーションサンプルは、単独でインキュベーション反応する細胞内エフェクター濃度相対の大きな増加があった。プロトコルはシンプルで、APで行われるのに十分に高速である病原性タンパク質の細胞内標的化および機能を含む、宿主細胞中の病原体タンパク質の高スループット特性評価を可能にarallelファッション。

概念の最初の証明として、精製された緑色蛍光タンパク質が正常にエレクトロポレーションを用いて哺乳動物細胞に導入した。 GFP、おおよそ27 kDの分子量のタンパク質は、一般的に重要な細胞毒性なし分子生物学のツールと​​して（通常は、プラスミドDNAから発現）哺乳動物細胞に導入される。 HeLa細胞（ 図1A）インキュベートまたは蛍光GFPシグナルをチェックするために、免...

Watch this Scientific Journal Video about Electroporation of Functional Bacterial Effectors into Mammalian Cells at JoVE.com

Electroporation of Functional Bacterial Effectors into Mammalian Cells

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

哺乳動物細胞への機能性細菌エフェクターのエレクトロポレーション

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

electroporation-of-functional-bacterial-effectors-into-mammalian-cells

Research

JoVE Journal

Immunology and Infection

10.0K ビュー.  Pacific Northwest National Laboratory. Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells. 

ビデオ: Electroporation of Functional Bacterial Effectors into Mammalian Cells

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

細菌性病原体によるヒト細胞の浸潤

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

免疫・感染学

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

哺乳類上皮細胞上に細菌付着のインビトロアッセイ

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

機能心臓の免疫細胞の単離

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

マダニScapularisダニの唾液、唾液腺、および体液コレクション

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

における骨髄細胞ダイナミクスのリアルタイムイメージング<em&gt;のApc<sup&gt;最小/ +</sup</em&gt;腸腫瘍ディスク共焦点顕微鏡スピニングすることにより

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

過ヨウ素酸シッフ染色で末梢血単核細胞中のグリコーゲンの検出

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

発生とのマルチ表現型ハイコンテンツスクリーニング<em&gt; Q熱リケッチア</em&gt;トランスポゾン変異体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

細菌人工染色体：プラス鎖RNAウイルスの研究のための機能ゲノミクスツール

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

哺乳動物宿主細胞に分泌された細菌毒素の影響を研究するために、透過膜を挿入ベースの感染システムの実現

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

酵母ツーハイブリッドスクリーンを用いた非耕作植物病原体から細菌のエフェクターの機能をUnravelling

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...