我々は、重要な実験的な評価と議論のためUICでオブライアンラボに感謝します。臨床およびトランスレーショナルサイエンスグラントUL1TR000050ためのUICセンター - この作品は、トランスレーショナル研究を進めるためのGKCとナショナルセンターに米国心臓協会グラント11SDG5230003によってサポートされていました。

1。細胞トランスフェクション<ol><li>プレート細胞トランスフェクションの前日に、免疫のために少数の細胞をプレーティングする。 <ol><li>免疫蛍光の場合：6ウェルプレート、コートガラスカバースリップ（1ウェルあたり）少なくとも4時間、37℃で0.02％ゼラチンで、培地で1回洗浄し、各ウェルのために、プレート1×10 5 HEK 293T 2ミリリットル完全増殖培地中の細胞[ダルベッコ改変イーグル培地（DMEM）を加えた10％FBS]。 </li><li> 6ウェルプレート（0.3×10 5 HEK 293T細胞を、24ウェルプレートのウェルあたり0.5ミリリットル完全増殖培地中で2ミリリットル完全増殖培地中でウェルプレート1.2×10 5 HEK 293T細胞/：フローサイトメトリーおよびウェスタンブロット分析のために）。 </li></ol></li><li>製造業者のプロトコールに従ってトランスフェクションのためにDNAを準備のEffectene（キアゲン）を免疫するために使用されます。通過LT1（MIRUS）をフローサイトメトリーおよびウェスタンブロット分析のために使用される。使用されるDNAの量を以下に示す。 <table border="1"><tbody><tr><tD&gt; </td><td> 24ウェルプレート（NG） </td><td> 6ウェルプレート（ngの） </td></tr><tr><td> CFP-ベクトル</td><td> 10 </td><td> 50 </td></tr><tr><td> VN N-AKAP-LBC </td><td> 200 </td><td> 1000年</td></tr><tr><td> VC N-PDE4D3-FL（フルレングスPDE4D3） </td><td> 2.5、5、10、20、40、80 </td><td> 100 </td></tr><tr><td> VC N-PDE4D3-UCR1（UCR1ドメインを含むPDE4D3 N末端 ​​断片） </td><td></td><td> 100 </td></tr><tr><td> VC N-PDE4D3-UCR2 + CAT（UCR2と触媒ドメインを含むPDE4D3 C末端断片） </td><td></td><td> 100 </td></tr></tbody></table>トランスフェクションのEffectene（6ウェル）を用いた。 <ol><li>プラスミドDNAの指示された量+ 100μlのBuffer EC〜4μlのエンハンサー、ミックスを追加し、室温で5分間インキュベートする。 </li><li> 5μLのEffectene、ミックスを追加し、10分でインキュベート室温。細胞に滴下追加。 </li></ol> TRANSIT-LT1を用いたトランスフェクション（6-well/24-well）： <ol><li>滅菌チューブ内のOpti-MEM I無血清培地の250μl/50μlにプラスミドDNAの指示量を追加します。 </li><li>穏やかに混合するために希釈したDNA混合物およびピペットに移行-LT1試薬を追加します。 （TRANSIT-LT1試薬：DNA比はDNA1μgの当たりTRANSIT-LT1試薬3μlのです）。 </li><li>室温で30分間インキュベートし、細胞に滴下加える。 </li></ol></li></ol> 2。フローサイトメトリー、ウェスタンブロットや免疫細胞のための準備<ol><li> 24時間トランスフェクション後、細胞を採取する前に、落射蛍光顕微鏡下で蛍光を確認してください。 </li><li>フローサイトメトリー解析のための細胞調製： <ol><li> 24-well/6-wellプレートは0.5ミリリットル/ 2ミリリットル氷冷PBSで細胞を洗浄します。 </li><li> 50μl/250μlの0.05％トリプシンを用いて細胞を切り離し。 </li><li> 200μL/ 1ミリリットルDMEM培地でトリプシンを中和。 </li><li>共同5分間500×gで遠心分離によってllect250μlの細胞（両方とも、24ウェルおよび6ウェルプレートの場合）、ウェスタンブロット分析のために残っている細胞（1ml）を使用する。 </li><li>フローサイトメトリー分析のための氷の上で250μlのFACSバッファー[PBS + 0.5％（w / v）のBSA + 2mMのEDTA]、店舗で再懸濁した細胞。フローサイトメトリー分析が即時ならない場合細胞もまた（PBS中）を1％ホルムアルデヒド中で固定することができる。 </li></ol></li><li>ウェスタンブロット分析のための細胞調製する：フローサイトメトリー分析のための細胞調製物を並行して行う。 <ol><li> 2 mlの氷冷PBSで1×細胞を洗浄、細胞溶解緩衝液[10mMリン酸ナトリウムバッファー、pH 6.95、150mMのNaCl、5mMのEDTA、5mMのEGTA、1％トリトンX-100]の100μLを添加して細胞を溶解する。 </li><li> 4℃で21,000×gで10分間遠心分離によって細胞溶解物を収集℃で</li><li> 、ブラッドフォードタンパク質アッセイによりタンパク質濃度を定量、SDS-PAGE用レーンあたり等量のタンパク質をロードし、イムノ用ニトロセルロースに移す。 </li></OL&gt; </li><li>免疫用細胞調製。 <ol><li> 2ミリリットルPBSで3倍の細胞を洗浄し、室温で10分間、3.7％パラホルムアルデヒド（PBS中）1mlに細胞を固定し、その後5分間2ミリリットルPBSで3回洗浄する。 </li><li>封入剤を使用して、スライドガラス上にスリップをカバーマウント。必要に応じて、マニキュアを使用してカバースリップの端をシールします。 </li><li> 4でスライドを保管°C検査の前に。 </li></ol></li></ol> 3。フローサイトメトリーおよび免疫蛍光顕微鏡<ol><li>フローサイトメトリーは488nmで、波長405nm、及び642 nmの固体レーザーを備えたベックマン·コールター（シアン）ADPを使用して行われる。サミットソフトウェアは、取得および分析のために使用されている。 <ol><li>標準ビーズを用いたフローサイトメーターのキャリブレーションを行います。 </li><li>ゲー生細胞集団のためのリニアスケールでプロット前方散乱（FSC）/脇散乱（SSC）。 </li><li>ゲー非集約生細胞集団のためにプロット電圧/ FSCパルス幅。 </li><li>プロットcount/FL6（CFP fluorescencゲー非集約ライブCFP-陽性細胞のログスケールでE、405 nm）を。 </li><li> BIFC強度のログスケールでプロットcount/FL1（YFP蛍光488 nm）を。 </li><li> FSC、SSC、FL1とFL6光電子増倍管（PMT）各信号のしきい値を設定するには、電圧を調整、YFP-CFP-ダブルネガティブサンプルを実行します。 </li><li>将来オフライン補償単一の正（両方YFP +とCFP +）サンプルを実行する</li><li>分析のために少なくとも10,000ゲーテッドイベント（非集約ライブCFP +細胞）を取得。 </li></ol></li><li>免疫蛍光顕微鏡ツァイスLSM 510共焦点顕微鏡を用いて行われる注：それは間のすべての設定（ズーム等、ピンホール、検出器利得は、増幅器のオフセット、フレームサイズ、走査速度、走査平均値、およびレーザパワー）一貫性を保つために重要であるすべてのデータの適切な比較のためのサンプル。 </li></ol> 4。データ解析（サミット·ソフトウェアを使用して実行） <ol><li> acquisitと同様の分析プロトコルを設定する適切なゲーティングのイオン： 生きた細胞のためのFSC / SSCドットプロットにゲート1。 非集約生細胞のゲート1のFSC /パルス幅でゲート2。 非集約CFP +生細胞のゲート3のカウント/ CFPでゲート3。 </li><li> YFP +とCFP +シングルポジティブ細胞を用いYFPとCFP信号を補償します。 </li><li> CFP / YFP陽性細胞に再決定するカットオフのライン。 </li><li>エクスポートYFPはCFPの蛍光強度（MFI）+データプロットするためのExcelへの細胞を意味する。 </li><li> AKAP-LBC、PDE4D3の発現レベルにYFP MFIを正規化し、ウェスタンブロットによって決定されるように、α-チューブリン制御を読み込む。 </li></ol>

<ol>
	<li>Collura, V., Boissy, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+protein-protein+complexes+to+interactomics.">From protein-protein complexes to interactomics.</a> Subcell Biochem. 43, 135-183 (2007).</li><li>Stumpf, M. P., 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=Estimating+the+size+of+the+human+interactome.">Estimating the size of the human interactome.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 6959-6964 (2008).</li><li>Shekhawat, S. S., Ghosh, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split-protein+systems:+beyond+binary+protein-protein+interactions.">Split-protein systems: beyond binary protein-protein interactions.</a> Curr. Opin. Chem. Biol. 15, 789-797 (2011).</li><li>Dwane, S., Kiely, 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=Tools+used+to+study+how+protein+complexes+are+assembled+in+signaling+cascades.">Tools used to study how protein complexes are assembled in signaling cascades.</a> Bioeng. Bugs. 2, 247-259 (2011).</li><li>Piehler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+methodologies+for+measuring+protein+interactions+in+vivo+and+in+vitro.">New methodologies for measuring protein interactions in vivo and in vitro.</a> Curr. Opin. Struct. Biol. 15, 4-14 (2005).</li><li>Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+implementation+of+bimolecular+fluorescence+complementation+(BiFC)+assays+for+the+visualization+of+protein+interactions+in+living+cells.">Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.</a> Nat. Protoc. 1, 1278-1286 (2006).</li><li>Shyu, Y. J., Suarez, C. D., Hu, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+ternary+complexes+in+living+cells+by+using+a+BiFC-based+FRET+assay.">Visualization of ternary complexes in living cells by using a BiFC-based FRET assay.</a> Nat. Protoc. 3, 1693-1702 (2008).</li><li>Wong, K. A., O'Bryan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+fluorescence+complementation.">Bimolecular fluorescence complementation.</a> J. Vis. Exp. (50), e2643 (2011).</li><li>Sugarbaker, E. V., Thornthwaite, J. T., Temple, W. T., Ketcham, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+general+principles+and+applications+to+selected+studies+in+tumor+biology.">Flow cytometry: general principles and applications to selected studies in tumor biology.</a> Int. Adv. Surg. Oncol. 2, 125-153 (1979).</li><li>Koshy, S., Alizadeh, P., Timchenko, L. T., Beeton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+GLUT4+translocation+to+the+plasma+membrane+by+flow+cytometry.">Quantitative measurement of GLUT4 translocation to the plasma membrane by flow cytometry.</a> J. Vis. Exp. (45), e2429 (2010).</li><li>Smith, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+confocal+microscopy.">Basic confocal microscopy.</a> Curr. Protoc. Mol. Biol. Chapter 14 (Unit 14), 11 (2008).</li><li>Westwick, J. K., Lamerdin, 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=Improving+drug+discovery+with+contextual+assays+and+cellular+systems+analysis.">Improving drug discovery with contextual assays and cellular systems analysis.</a> Methods Mol. Biol. 756, 61-73 (2011).</li><li>Kilpatrick, L. E., Holliday, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+pharmacology+of+G+protein-coupled+receptor+signaling+complexes+using+bimolecular+fluorescence+complementation.">Dissecting the pharmacology of G protein-coupled receptor signaling complexes using bimolecular fluorescence complementation.</a> Methods Mol. Biol. 897, 109-138 (2012).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

BIFCは、タンパク質 - タンパク質相互作用を研究するためのシンプルかつ高感度な方法である。この方法は、新しいタンパク質 - タンパク質相互作用を識別するために使用することができない、しかし、それは細胞内タンパク質 - タンパク質相互作用を確認することと機能的特性を研究するために特に便利である等のタンパク質複合体の細胞内局在として、タンパク質 - タンパク質相互作用?...

65万タンパク質-タンパク質相互作用は、正常な細胞機能1,2を維持するために重要な役割を果たし、ヒトインタラクに存在することが推定される。他にも共免疫沈降（共IP）、細胞溶解物からのタンパク質-タンパク質相互作用を研究するための金本位、いくつかのタンパク質断片相補アッセイ（PCA）は、セル3の内部タンパク質-タンパク質相互作用の検出感度を向上させるために開発されてきた。技術はフェルスター共鳴エネルギー移動（FRET）、生物発光共鳴エネルギー移動（BRET）、および二分子蛍光相補（BIFC）4,5が含まれています。それぞれの潜在的な相互作用タンパク質パートナー（この例では、我々はAKAP-LBC及びPDE4D3を使用してください）​​に融合されています。BIFCは蛍光タンパク質（YFP変異体ここでは金星を使用します）の二つの断片の促進協会に基づいています。 fで可視化することができる機能的な蛍光の細胞結果内部の関心の二つのタンパク質の相互作用ライブまたは固定された細胞の6,7,8を使用luorescence顕微鏡。他のPCAに比べ、BIFCは、生きた細胞で細胞内局在を検討する可能性のある、高感度かつ少ない技術的に困難である。この技術の主な欠点は、しかしながら、一度形成された蛍光タンパク質複合体が逆転することができないことである。したがって、動的なタンパク質 - タンパク質相互作用を研究するための優れた方法ではない。本稿では、金星の蛍光強度をモニターすることによってAKAP-LBCでPDE4D3の相互作用部位をマッピングするBIFC使用。 （自動ハイスループット機械を用いて行われていない場合）、時間がかかり、労働集約的である蛍光顕微鏡を用いた従来の分析と比較して、フローサイトメトリーは、短時間9上で不均一な集団における細胞の数千の簡単な定量的な分析を提供10。ここで、フローサイトメトリー分析 - BIFCおよび共IP伝統的なサイドバイサイドの比較を行うことにより、我々は、2メートル示すethodsは、比較可能なデータを提供するが、フローサイトメトリー分析は、BIFC少ない時間がかかり、目的の細胞に限られている場合に、より有用であり得る少ない材料を使用する。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Dulbecco's modified Eagle's media (DMEM)</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin (pen/strep), 100x</td>
			<td>Invitrogen</td>
			<td>15070-063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-Flag antibody</td>
			<td>Sigma</td>
			<td>M2, F1804</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-HA antibody</td>
			<td>Covance</td>
			<td>16B12, MMS-101R</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&alpha;-tubulin</td>
			<td>Sigma</td>
			<td>DM1A, T9026</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Clontech</td>
			<td>632569</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell culture plates, 6-well tissue culture treated</td>
			<td>Thermo Fisher Scientific</td>
			<td>130184</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEK 293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Maxiprep plasmid purification kit, high speed</td>
			<td>Qiagen</td>
			<td>12663</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Phosphate-buffered saline (DPBS), sterile 1x</td>
			<td>Invitrogen</td>
			<td>14190-144</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.05% (w/v)</td>
			<td>Gibco</td>
			<td>25300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polystyrene round-bottom tubes for FACS staining</td>
			<td>BD Biosciences</td>
			<td>352052</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>S74337MF</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Prolong gold antifade reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P-36931</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superfrost plus Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fisherfinest premium cover glass</td>
			<td>Fisher Scientific</td>
			<td>12-548-5P</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>CO2 air-jacketed Incubator</td>
			<td>NuAIR DH autoflow</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Confocal microscope LSM510 META</td>
			<td>Carl Zeiss, Inc</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electrophoresis and transfer unit</td>
			<td>Biorad</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cyan ADP Flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

flow cytometric analysis of bimolecular fluorescence complementation: a high throughput quantitative method to study protein-protein interaction

細胞内のタンパク質 - タンパク質相互作用を研究するための方法のうち、二分子蛍光相補（BIFC）は比較的簡単と小文字が区別されます。 BIFCは、蛍光タンパク質の二非蛍光フラグメント（金星、黄色蛍光タンパク質の変異体は、ここで使用されている）を使用した蛍光の生産に基づいている。非蛍光金星断片（VNとVC）が原因でVN-AKAP-LBC-VC-PDE4D3相互作用と機能的蛍光タンパク質の形成に蛍光降伏、2相互作用するタンパク質（この場合は、AKAP-LBC及びPDE4D3）に融合されている細胞内。 BIFCは、タンパク質複合体の細胞内局在と蛍光強度に基づいてタンパク質相互作用の強さについての情報を提供します。しかしながら、タンパク質 - タンパク質相互作用の強さを定量化するために顕微鏡を用いBIFC解析には時間がかかり、タンパク質発現と相互作用における不均一性に起因幾分主観的である。フローサイトメトリーを結合することによってBIFC方法論分析は、蛍光BIFCタンパク質 - タンパク質相互作用信号は、正確に短時間で細胞の大量のために測定することができる。ここでは、AKAP-LBCとの相互作用に必要とされるPDE4D3でマップ地域にこの方法論の適用を実証する。このハイスループット法は、タンパク質 - タンパク質相互作用を調節する因子のスクリーニングに適用することができる。

AKAP-LBCとPDE4D3構築物（ 図1B）はそれぞれ、VNおよびVC断片と融合させた。 AKAP-LBCと機能YFP蛍光（ 図1A）でPDE4D3結果の相互作用。ここでは、PDE4D3でAKAP-LBC-結合部位をマッピングするBIFCメソッドを使用します。上流に保存された領域1（UCR1）、上流の保存された領域2（UCR2）と触媒領域（CAT）PDE4D3の結合部位をスクリーニングするために使用されたため、完全長（FL）、UCR1...

Watch this Scientific Journal Video about Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction at JoVE.com

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

二分子蛍光相補のフローサイトメトリー分析は、タンパク質 - タンパク質相互作用を研究するためのハイスループット定量方法を提供するものである。この方法は、マッピング·タンパク質結合部位およびタンパク質 - タンパク質相互作用を調節する因子をスクリーニングするために適用することができる。

二分子蛍光相補のフローサイトメトリー分析：タンパク質 - タンパク質相互作用を研究するためのハイスループット定量法

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

flow-cytometric-analysis-bimolecular-fluorescence-complementation

Research

JoVE Journal

Biology

18.3K ビュー.  University of Illinois at Chicago. 二分子蛍光相補のフローサイトメトリー分析は、タンパク質 - タンパク質相互作用を研究するためのハイスループット定量方法を提供するものである。この方法は、マッピング·タンパク質結合部位およびタンパク質 - タンパク質相互作用を調節する因子をスクリーニングするために適用することができる。

ビデオ: Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

神経毒性の高いコンテンツ分析のための神経とアストロサイト共培養アッセイ

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

生物学

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

グローバルS. cerevisiaeの細胞小器官の形態を研究するためにハイスループット法

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

ヘリオス遺伝子銃を用いたタマネギの細胞における蛋白質 - 蛋白質相互作用のための二分子蛍光相補（BiFC）アッセイ

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

二分子蛍光相補

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

ゲートウェイ互換二分子蛍光相補（BiFC）システムを使用してプラントにおけるタンパク質相互作用の検出

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

タンパク質 - タンパク質相互作用を研究するために磁気ビーズを用いて液相アフィニティキャプチャーアッセイ：ポリオ-nanobodyの例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

高度な3D蛍光顕微鏡を用いたオートファジーの定量分析

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

ペプチドエピトープの定量分析のための高スループット、MHC II結合アッセイ

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

タバコプロトプラストと葉で二分子蛍光相補性によって可視化し、タンパク質間相互作用

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

腫瘍球の高スループット画像解析：自動的かつ正確にスフェロイドのサイズを測定するための使いやすいソフトウェアアプリケーション

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...