著者は、技術支援のSLOプラスミドとジョナサンフランクスの寛大な贈り物のためにanthrolysin O、マイケルCaparonの寛大な贈り物をリチャードレストに感謝したいと思います。この作品は、NIHの助成T32CA82084（PAK）とR01AI072083（RDS）によって賄われていた。

1。ストレプトリジンO（SLO）の精製<ol><li> 0.5LのLBブロスにプラスミドpBADgIII SLOhis-9を含むBL21 GOLDの細胞を20mlの一晩培養物を接種し、500μlの50 mg / mlのアンピシリンを追加します。 37℃で225rpmで振とう培養し、OD 600 = 0.6になるまでには、通常1.5時間を〜。 10,000 5分xgで1 mlの細菌（T 0と見なされます）遠心し、140μlの1×SDSサンプルバッファー/ 1 ODでペレットを溶解し、タンパク質の純度分析用せん断DNAに超音波洗浄します。 </li><li>文化〜5mlの20％のアラビノースを添加して細菌を誘導し、3時間室温で225rpmで振とうする。細菌の1ミリリットルを収集し、純度分析用のT 3の時点について、上記の1×SDSサンプルバッファーで遠心し、溶解する。 </li><li> 500mlの遠心ボトルに残っている細菌を収集し、4℃（8500回転ソーバルGS-3ローター）で12分間、12,000×gで遠心する。必要であれば℃で一晩以上-80℃で上清、店舗ペレットをデカントします。 </li><li> frを再懸濁400μlの25パーセントのトリトンX-100、100μlの100 mg / mlのリゾチーム、50で補足10ミリリットル/洗浄溶解緩衝液（50mMのNaH 2 PO 4、300mMのNaCl、10mMイミダゾール、pH8.0）中で氷上ozenペレットμlの200 mMのフッ化フェニルメチルスルホニルフルオリド（PMSF）。 50ミリリットルオークリッジ管に再懸濁ペレットを転送します。 </li><li> 30秒間隔で、氷上で30秒間5回ライセートを超音波洗浄します。スピンは4℃（18,000 rpmのソーバルSS-34ローター）で20分間39000×gでオークリッジ管で溶解液を超音波処理した。 </li><li>ライセートが回転しているときに、4℃で5分間1200 rpmで洗う/溶解バッファーとスピン10mlで1ミリリットルNi-NTAアガロースを洗浄℃に（それ以降のすべての洗浄/溶出は、これらの条件下で遠心分離を使用します）。洗浄を吸引します。 </li><li>ステップ1.5からNi-NTAアガロースに細菌の上清を追加し、4℃で2.5時間穏やかに振とう℃、上清を回収するためにスピン。 10μ4X SDSサンプルバッファーで上清30μlを組み合わせると純度分析のために保存。洗浄/溶解緩衝液中で、オンビーズを4回洗うトリス/塩緩衝液（50mM Tris、300mMのNaCl、pH8.0）にCE。 </li><li> 、ビーズに1 mlの溶出バッファー（トリス/塩緩衝液中の250mMイミダゾール）および5μlの1Mのジチオスレイトール（DTT）を添加すること、氷上で10分間インキュベートし、紡績、上清を収集することにより、3回溶出する。 SLOは非常にレドックス感受性タンパク質であり、常に氷上に維持しなければなりません。でも、時間の短い期間に、37℃に加温した場合、一度減少し、タンパク質は急速にその活性を失うことになる。 </li><li>溶出緩衝液で500μlのポリミキシン共役アガロース、4℃で1分を500μlのCを再懸濁し、10,000 xgでスピンを洗う。エンドトキシンを枯渇させるために、それぞれの溶出に200μlのビーズを追加して、30分間、4℃を振る。 1 4時分℃、上清を保存し、10,000 xgでスピン。 SDS-PAGE解析のために、10μlの4X SDSサンプルバッファーで各溶出液30μlを兼ね備えています。 </li><li> SDS-PAGEによる溶出の純度をテストします。沸騰サンプルは5分間95℃でゲル解析するために保存し、10％ゲルで10μlをロードします。 GEを染色蛋白質を可視化するクマシー付きL（ 図2A）。 SLOは69 kDaである。 （典型的な収率は、最初の2つの溶出のために4 mg / mlのですが、使用する細菌株およびプラスミドによって異なる場合があります）タンパク質濃度を決定するために、ブラッドフォードアッセイを行う。 </li><li>それぞれの溶出の溶血活性をテストします（下記参照）。純度、濃度、溶血活性が良好であるプールの溶出。シングルユースのアリコート（通常5〜10μL）にアリコートSLOは、-80℃でドライアイスと℃で保存時にフリーズ</li></ol> 2。溶血アッセイ<ol><li> RBCアッセイ緩衝液（0.3％ウシ血清アルブミン（BSA）、2mMのCaCl 2、10mMのHepes、pH7.4）で5分間1200 rpmでスピンでヒツジ赤血球（RBC）を洗浄し、2.5％の赤血球における懸濁する10ミリリットルのRBCアッセイバッファー。 </li><li> /ウェル氷上で96ウェルV底プレートに10μlのRBCのアッセイバッファーを追加します。直列二連で各溶出を薄める。典型的な希釈範囲は1:512,000に1:1000である。 10でない毒素を持つ3つのウェルと3つのウェルを含むそれぞれ、最小と最大の井戸としてTriton X-100を2パーセントずつ。 </li><li> 37℃で30分間インキュベートし、プレートをカバーしています。 5分間1,200 rpmで遠心分離します。平底96ウェルプレートに上清70μlを。 405を読んでください。 1単位は、RBCの50％溶解するために必要な毒素の希釈である。毒素活性を1ユニット*希釈factor/0.01 mlである。 </li></ol> 3。細胞溶解アッセイ<ol><li>細胞を回収し、カウント、スピン。 2×10 6バッファR / B（2mMのCaCl 2を含むRPMI、0.5％BSA）および20μg/ mlのヨウ化プロピジウムで/ mlで再懸濁する。 96ウェルV底プレートに100μlの細胞を追加します。 </li><li>シリアルバッファR / Bで最終濃度が2倍にSLOを希釈し、細胞に100μlの毒素または100μlバッファR / Bを追加します。 5分間37℃でインキュベートするU / mlの2000から31.25 U / mlの最終濃度に典型的な範囲です。 </li><li>フローサイトメーター上でセルを実行し、フィコエリトリン（PE）のためのフィルタを使用してデータを収集します。 1ログシフトは一過透過処理セルを表しますLS 3ログシフトは死んだ細胞4を示している。次のように、実験（％PIの高い EXP）からの制御で死んだ細胞の割合（％PIの高い CTL）を差し引くことにより、細胞の特異的溶解を計算する：特異的溶解=（％PIの高い EXP - ％PIの高い CTL） /（100 - ％PIの高い CTL）* 100 </li></ol> 4。培養皿で細胞への毒素のマイクロピペットデリバリー<ol><li>コラーゲンコートガラスボトム35mmディッシュの実験は、プレート2×10 5のマクロファージへの1日前。 </li><li>顕微鏡やマイクロインジェクターをオンにします。加熱ステージ時間は37℃に温め許可顕微鏡の選択は、通常、ローカルで使用可能であるかに依存します。顕微鏡は、倒立ステージ37に選ばれた染料と、物理的に微量注入器を接続するためのスペースに適し℃、励起/蛍光フィルターキューブを皿を加熱することができる段階を必要とします。バートランドレンズマイクロインジェクションのために役立ちますが、必須ではありません。顕微鏡を駆動するコンピュータは、データを収集し、保存するための十分なメモリが必要です。 </li><li> 37℃染料で30分間標識細胞℃、アッセイに応じて、標識は、1ミリリットルのフルメディアにAM 1 mlのPBSまたは2μlのカルセインで午前5μlのFura2で行うことができる。 Fura2は、励起/発光は510分の340 nmと510分の380 nmのために収集され、380分の340の信号の比は、カルシウムフラックスを決定します。エチジウムホモは620分の525 nmで、APCは660分の650 nmである一方カルセインは、励起/発光は515分の495 nmである。他のラベルも同様に選択することができる。 </li><li> 2mMのCaCl 2を補充した1ミリリットルRPMIにPBSおよび場所で細胞を洗浄。顕微鏡にマウントします。 </li><li> 4℃、20,000×gで10分で毒素およびデキストラン水中で、遠心分離を薄める典型的には、1μlのSLOと4μlの10 mg / mlのデキストラン-555は6μlの水で希釈する。デキストランまたは別の蛍光液相分子は、フェムト先端が詰まっていないことを確認するために使用されており、麟蹄CTSは、必要に応じて。 </li><li> microloaderを使用して希釈された毒素の0.07μlの背面からフェムト先端をロードします。 </li><li>マイクロインジェクターにフェムト先端をロードします。先端はすべての方向に移動する部屋を有する細胞の中心上に座りますようにインジェクタの角度を調整します。 z軸の制限をクリアします。注入の設定は20psiの背圧は120 psiで0.5秒間注入する必要があります。それは、媒体に入るまで先端を下げる。 </li><li>バートランドレンズ、中央先端を使用して、それを細胞に近い下げられるように先端に従ってください。一度フォーカスを失い、通常の光学系に切り替えます。針の影は、フィールドには明らかであろう。セルの上でピントを合わせ、それが焦点になるまで、ニードルを下げてください。細胞上にバックフォーカスと慎重にセルに隣接する針を持参。注射用のz-limitを設定します。所望の位置に先端を移動し、撮影を開始し、所望の時点で毒素を解放するために注入する。針を上げ、細胞の新しい領域に移動して注入します。望ましくないことを防止するためにホームポジションに針を動かす針から毒素漏れ。 </li></ol>

<ol>
	<li>Walev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+proteins+into+living+cells+by+reversible+membrane+permeabilization+with+streptolysin-O.">Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 3185-3190 (2001).</li><li>Walev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+regulates+IL-1+beta+processing+via+calcium-independent+phospholipase+A2.">Potassium regulates IL-1 beta processing via calcium-independent phospholipase A2.</a> J. Immunol. 164, 5120-5124 (2000).</li><li>Scolding, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+removal+by+oligodendrocytes+of+membrane+attack+complexes+formed+by+activated+complement.">Vesicular removal by oligodendrocytes of membrane attack complexes formed by activated complement.</a> Nature. 339, 620-622 (1989).</li><li>Keyel, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptolysin+O+clearance+through+sequestration+into+blebs+that+bud+passively+from+the+plasma+membrane.">Streptolysin O clearance through sequestration into blebs that bud passively from the plasma membrane.</a> J. Cell. Sci. 124, 2414-2423 (2011).</li><li>MacKenzie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+secretion+of+interleukin-1beta+by+microvesicle+shedding.">Rapid secretion of interleukin-1beta by microvesicle shedding.</a> Immunity. 15, 825-835 (2001).</li><li>Qu, Y., Franchi, L., Nunez, G., Dubyak, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclassical+IL-1+beta+secretion+stimulated+by+P2X7+receptors+is+dependent+on+inflammasome+activation+and+correlated+with+exosome+release+in+murine+macrophages.">Nonclassical IL-1 beta secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages.</a> J. Immunol. 179, 1913-1925 (2007).</li><li>Andrei, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipases+C+and+A2+control+lysosome-mediated+IL-1+beta+secretion:+Implications+for+inflammatory+processes.">Phospholipases C and A2 control lysosome-mediated IL-1 beta secretion: Implications for inflammatory processes.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 9745-9750 (2004).</li><li>Franchi, L., Eigenbrod, T., Munoz-Planillo, R., Nunez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammasome:+a+caspase-1-activation+platform+that+regulates+immune+responses+and+disease+pathogenesis.">The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis.</a> Nat. Immunol. 10, 241-247 (2009).</li><li>Magassa, N., Chandrasekaran, S., Caparon, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+pyogenes+cytolysin-mediated+translocation+does+not+require+pore+formation+by+streptolysin+O.">Streptococcus pyogenes cytolysin-mediated translocation does not require pore formation by streptolysin O.</a> EMBO Rep. 11, 400-405 (2010).</li><li>Pinkney, M., Beachey, E., Kehoe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thiol-activated+toxin+streptolysin+O+does+not+require+a+thiol+group+for+cytolytic+activity.">The thiol-activated toxin streptolysin O does not require a thiol group for cytolytic activity.</a> Infect. Immun. 57, 2553-2558 (1989).</li><li>Watkins, S. C., Salter, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+connectivity+between+immune+cells+mediated+by+tunneling+nanotubules.">Functional connectivity between immune cells mediated by tunneling nanotubules.</a> Immunity. 23, 309-318 (2005).</li><li>McNeil, P. L., Vogel, S. S., Miyake, K., Terasaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patching+plasma+membrane+disruptions+with+cytoplasmic+membrane.">Patching plasma membrane disruptions with cytoplasmic membrane.</a> J. Cell. Sci. 113, 1891-1902 (2000).</li><li>Shannon, J. G., Ross, C. L., Koehler, T. M., Rest, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+anthrolysin+O,+the+Bacillus+anthracis+cholesterol-dependent+cytolysin.">Characterization of anthrolysin O, the Bacillus anthracis cholesterol-dependent cytolysin.</a> Infect. Immun. 71, 3183-3189 (2003).</li></ol>

特別な利害関係は宣言されません。

技術は細菌毒素に対する免疫細胞の反応の検査を可能にする、ここで説明。最も重要なステップは、毒素の取り扱いと投薬です。毒素活性はさらにそのもろさのために、同じ製剤の異なるアリコートの間、非常に可変にすることができます。これは、いずれかの基準セルラインや赤血球に対する毒素の各アリコートをテストする、または毒素のグラデーションを使用する必要が生じて。毒素?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td> Ni-NTAアガロース</td><td>キアゲン</td><td> 30210 </td><td></td></tr><tr><td>ポリミキシン-アガロース</td><td>シグマ</td><td> P1411-5ML </td><td></td></tr><tr><td> Zeba脱塩スピンCOL </td><td>フィッシャー</td><td> PI-89891 </td><td></td></tr><tr><td>ヒツジ赤血球</td><td>フィッシャー</td><td> 50-415-688 </td><td></td></tr><tr><td> pBADgIII-SLO </td><td> N / A </td><td> N / A </td><td> refは9を参照してください</td></tr><tr><td> Cy5の単反応性染料</td><td> GEヘルスケア</td><td> PA25001 </td><td></td></tr><tr><td> Fura2-AM </td><td>ライフテクノロジーズ</td><td> F1221 </td><td></td></tr><tr><td>カルセインAM / EThidiumホモダイマー</td><td>ライフテクノロジーズ</td><td> L3224 </td><td></td></tr><tr><td>抗CD11c-APC </td><td> BD Biosciences社</td><td> 550261 </td><td></td></tr><tr><td>コラーゲンコートガラスボトムディッシュ</td><td>マテック</td><td> P35GCol-1.5-10-C </td><td></td></tr><tr><td>フェムト先端II </td><td>フィッシャー</td><td> E5242957000 </td><td></td></tr><tr><td> Microloader </td><td>フィッシャー</td><td> E5242956003 </td><td></td></tr><tr><td>デキストランアレクサ555 </td><td>ライフテクノロジーズ</td><td> D34679 </td><td></td></tr><tr><td> Injectman NI 2 </td><td>エッペンドルフ</td><td> 920000029 </td><td></td></tr><tr><td> FemtoJet </td><td>エッペンドルフ</td><td> 5247 000.013 </td><td></td></tr><tr><td></td><td></td><td></td><td>特定の試薬 ​​および設備を導入の表1。リストとソースENTが必要。会社とカタログ番号と一緒に、このプロトコルで使用される特定の機器や試薬は、記載されています。 </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> バッファ </td><td> 構図 </td><td> 使用されるステップ </td></tr><tr><td>溶解/洗浄</td><td>の50mMのNaH 2 PO 4 300mMのNaCl 10mMイミダゾール、pH8.0の</td><td> 1.4 </td></tr><tr><td>トリス/塩</td><td> 50mMのトリス、pH8.0 300mMのNaCl </td><td> 1.7 </td></tr><tr><td>溶出緩衝液</td><td> 50mMのトリス、pH8.0 300mMのNaCl 250mMイミダゾール</td><td> 1.8 </td></tr><tr><td> RBCアッセイバッファー</td><td> 0.3パーセントのBSA 2mMのCaCl 2 10mMのHEPES、pH7.4の</td><td> 2.1 </td></tr><tr><td>バッファR / B </td><td> RPMI培地細胞培養培地 2mMのCaCl 2 0.5％BSA </td><tD&gt; 3.1 </td></tr></tbody></table></td></tr><tr><td></td><td></td><td></td><td>このプロトコルで使用されるバッファの表2。一覧。使用されるバッファは、その組成とは、プロトコルで使用されるときの最初のステップが記載されています。 </td></tr></tbody></table>

visualization of bacterial toxin induced responses using live cell fluorescence microscopy

細菌毒素は、細胞内容物と外部環境からの物質の流入の漏洩を可能に孔を形成し、膜中のコレステロールと結合する。セルは、アクティブ膜修復プロセスを必要とするこの侮辱から回復、または他の毒素曝露や細胞タイプ1の量に応じて、死ぬことができます。さらに、これらの毒素は、炎症性サイトカインの配列2を産生するマクロファージなどの免疫細胞の活性化を介して感染したホスト内の強い炎症反応を誘導する。多くのグラム陽性菌は、主に特徴づけられていないメカニズムを介してそれらの病原性に寄与することが示されているコレステロール結合毒素を産生する。 これらの毒素に曝露した細胞の形質膜における形態学的変化は本質的な細胞防御を示唆し、細胞外空間に流すことができ、コレステロールに富んだ表面突起、に彼らの隔離を含む機構3,4。このプロセスは、代謝活性の不在下で、すべてのセルで発生し、化学的固定4の後にEMを使用して可視化することができる。そのような毒素曝露に応答して炎症を媒介マクロファージなどの免疫細胞は、誘導される膜小胞はIL-1ファミリーのサイトカインを含むことが示唆されており、毒素を流し、これらの炎症性サイトカイン5,6,7を広めるための両方の責任があるかもしれません。両方とも8カスパーゼ1依存プロセスであるとして、IL-1β放出および細胞死の特定の種類の間にリンクと呼ばれるpyroptosisが、示唆されている。膜小胞の毒素結合、脱粒、サイトカイン放出、および潜在的に細胞死を含むこのマクロファージ応答の複雑さを整理するために、我々には、毒素 - 細胞間の相互作用のリアルタイム可視化を可能にラベリング手法や蛍光顕微鏡法を開発した機能不全と死の測定（ 図1）。使用ライブセルイメージングの他の技術の制限のために必要です。フローサイトメトリーは、個々の細胞の高分解能、リアルタイム可視化を提供していないながら、生化学的アプローチは、個々の細胞で発生する効果を解決することはできません。ここで説明する方法は、細胞内の複雑な表現型の変化を伴うその他の刺激によって誘導される応答の動態解析に適用することができます。

典型的には10 7〜10 8 U / mlのSLOは4 mg / mlのタンパク質濃度を求めることができる。細胞溶解に必要な毒素の量は、細胞の種類によって異なりますが、通常125から500 U / mlのSLO（ 図2B）です。他人（特にT細胞株）は、より敏感であるけれどもマクロファージ様細胞型（4000 U / ml）を、より耐性があることができます。これらの感応度は市販のSLOに対応しています。毒素活性は?...

Watch this Scientific Journal Video about Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy at JoVE.com

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

組換え体からコレステロール結合毒素ストレプトリジンOの精製方法<em&gt; E大腸菌の</em真核細胞を生きるためにバインド毒素の&gt;と可視化が記載されている。毒素の局所送達は、毒素の生物学の新たな側面を明らかに標的細胞における迅速かつ複雑な変化を誘導する。

ライブセル蛍光顕微鏡を用いた細菌毒素誘導される応答の可視化

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

visualization-bacterial-toxin-induced-responses-using-live-cell

Research

JoVE Journal

Immunology and Infection

11.7K ビュー.  University of Pittsburgh School of Medicine. 組換え体からコレステロール結合毒素ストレプトリジンOの精製方法 E大腸菌のと可視化が記載されている。毒素の局所送達は、毒素の生物学の新たな側面を明らかに標的細胞における迅速かつ複雑な変化を誘導する。

ビデオ: Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

HIVとライブ共焦点顕微鏡の蛍光クローンを使用して、HIVの可視化細胞間伝達

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

免疫・感染学

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

全葉のイメージングを使用して細菌細胞をライブに気孔反応を評価する

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

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

の生細胞イメージング枯草菌と肺炎球菌自動タイムラプス顕微鏡を使用して

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

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

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

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

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

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

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

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

真菌病原体の食作用の生細胞ビデオ顕微鏡

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Application of Long-term cultured Interferon-&amp;#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

牛にエフェクターおよびメモリーT細胞応答を評価するための長期培養インターフェロンγ酵素結合免疫アッセイの応用

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

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 ...

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

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

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

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

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

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

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

単一細胞の蛍光顕微鏡による Efferocytosis の定量化

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

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.