discovering-protein-interactions-characterizing-protein-function

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...

Promega Corporation

A method to simultaneously determine the relative numbers of live and dead cells in culture by introducing a combination of two fluorogenic substrates or a fluorogenic and a luminogenic protease substrate into the sample is described. The method is based on detection of differential ubiquitous proteolytic activities associated with intact viable cells and cells that have lost membrane integrity. A cell-permeable peptide aminofluorocoumarin substrate detects protease activity restricted to intact viable cells. Upon cell death, the viable cell protease marker becomes inactive. An impermeable peptide rhodamine 110 (or aminoluciferin) conjugated substrate detects protease activity from nonviable cells that have lost membrane integrity. The multiplex assay can detect 200 dead cells in a population of 10,000 viable cells. The protease substrate reagents do not damage viable cells over the course of the assay, thus the method can be multiplexed further with other assays in a homogeneous format. Ratiometric measurement of viable and dead cells in the same sample provides an internal control that can be used to normalize data from other cell-based assays.

A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers.

&#x4ECB;&#x7ECD;&#x4E00;&#x79CD;&#x65B9;&#x6CD5;&#xFF0C;&#x540C;&#x65F6;&#x901A;&#x8FC7;&#x5F15;&#x5165;&#x6837;&#x54C1;&#x7684;&#x4E24;&#x4E2A;&#x8367;&#x5149;&#x57FA;&#x677F;&#x6216;&#x8367;&#x5149;&#x548C; luminogenic &#x86CB;&#x767D;&#x9176;&#x5E95;&#x7269;&#x7684;&#x7EC4;&#x5408;&#x6765;&#x786E;&#x5B9A;&#x7684;&#x6587;&#x5316;&#x751F;&#x6D3B;&#x548C;&#x6B7B;&#x7EC6;&#x80DE;&#x7684;&#x76F8;&#x5BF9;&#x6570;&#x91CF;&#x3002;&#x8BE5;&#x65B9;&#x6CD5;&#x57FA;&#x4E8E;&#x5FAE;&#x5206;&#x65E0;&#x5904;&#x4E0D;&#x5728;&#x86CB;&#x767D;&#x6C34;&#x89E3;&#x4E0E;&#x76F8;&#x5173;&#x7684;&#x6D3B;&#x52A8;&#x5B8C;&#x6574;&#x53EF;&#x884C;&#x7684;&#x5355;&#x5143;&#x683C;&#x548C;&#x5355;&#x5143;&#x683C;&#x5931;&#x53BB;&#x4E86;&#x819C;&#x5B8C;&#x6574;&#x6027;&#x68C0;&#x6D4B;&#x3002;&#x7EC6;&#x80DE;&#x7A7F;&#x900F;&#x80BD; aminofluorocoumarin &#x57FA;&#x677F;&#x68C0;&#x6D4B;&#x86CB;&#x767D;&#x9176;&#x6D3B;&#x6027;&#x9650;&#x4E8E;&#x5B8C;&#x6574;&#x53EF;&#x884C;&#x7684;&#x5355;&#x5143;&#x683C;&#x3002;&#x7EC6;&#x80DE;&#x6B7B;&#x4EA1;&#x540E;&#x53EF;&#x884C;&#x7684;&#x7EC6;&#x80DE;&#x86CB;&#x767D;&#x9176;&#x6807;&#x8BB0;&#x53D8;&#x4E3A;&#x975E;&#x6D3B;&#x52A8;&#x72B6;&#x6001;&#x3002;&#x9632;&#x6E17;&#x80BD;&#x7F57;&#x4E39; 110 &#xFF08;&#x6216; aminoluciferin&#xFF09; &#x5171;&#x8F6D;&#x7684;&#x57FA;&#x677F;&#x68C0;&#x6D4B;&#x86CB;&#x767D;&#x9176;&#x6D3B;&#x6027;&#x4ECE;&#x8D70;&#x4E0B;&#x5761;&#x7684;&#x5355;&#x5143;&#x683C;&#x5931;&#x53BB;&#x4E86;&#x819C;&#x5B8C;&#x6574;&#x6027;&#x3002;&#x591A;&#x91CD;&#x68C0;&#x6D4B;&#x53EF;&#x4EE5;&#x68C0;&#x6D4B; 200 &#x6B7B;&#x7EC6;&#x80DE;&#x4E2D;&#x4EBA;&#x53E3;&#x7684; 10000 &#x4E2A;&#x53EF;&#x884C;&#x7684;&#x5355;&#x5143;&#x3002;&#x86CB;&#x767D;&#x9176;&#x5E95;&#x7269;&#x8BD5;&#x5242;&#x6D4B;&#x5B9A;&#x63A8;&#x79FB;&#x4E0D;&#x635F;&#x4F24;&#x53EF;&#x884C;&#x7684;&#x5355;&#x5143;&#x683C;&#xFF0C;&#x56E0;&#x6B64;&#x53EF;&#x4EE5;&#x590D;&#x7528;&#x65B9;&#x6CD5;&#x8FDB;&#x4E00;&#x6B65;&#x4E0E;&#x5176;&#x4ED6;&#x5316;&#x9A8C;&#x5747;&#x8D28;&#x7684;&#x683C;&#x5F0F;&#x3002;&#x540C;&#x4E00;&#x6837;&#x54C1;&#x4E2D;&#x7684;&#x53EF;&#x884C;&#x548C;&#x6B7B;&#x7EC6;&#x80DE;&#x6BD4;&#x7387;&#x8BA1;&#x91CF;&#x6D4B;&#x91CF;&#x63D0;&#x4F9B;&#x4E86;&#x53EF;&#x7528;&#x4E8E;&#x89C4;&#x8303;&#x5316;&#x6570;&#x636E;&#x4ECE;&#x5176;&#x4ED6;&#x57FA;&#x4E8E;&#x7EC6;&#x80DE;&#x68C0;&#x5B9A;&#x7684;&#x5185;&#x90E8;&#x63A7;&#x5236;&#x3002;

&#x6D4B;&#x91CF;&#x540C;&#x4E00;&#x6837;&#x54C1;&#x4E2D;&#x7684;&#x6D3B;&#x7684;&#x548C;&#x6B7B;&#x7EC6;&#x80DE;&#x7684;&#x4E0D;&#x540C;&#x86CB;&#x767D;&#x9176;&#x6807;&#x5FD7;&#x68C0;&#x6D4B;&#x5747;&#x76F8;&#x6D4B;&#x5B9A;&#x3002;

On d&eacute;crit une m&eacute;thode pour en m&ecirc;me temps d&eacute;terminer le nombre relatif de cellules vivantes et mortes en culture en introduisant une combinaison de deux substrats fluorog&eacute;niques ou un fluorog&eacute;niques et un substrat de prot&eacute;ase LUMINOG&Egrave;NE dans l&#39;&eacute;chantillon. La m&eacute;thode est bas&eacute;e sur la d&eacute;tection des diff&eacute;rentielles activit&eacute;s prot&eacute;olytiques omnipr&eacute;sentes associ&eacute;es &agrave; des cellules viables intactes et les cellules qui ont perdu l&#39;int&eacute;grit&eacute; de la membrane. Un substrat d&#39;aminofluorocoumarin peptide perm&eacute;able &agrave; la cellule d&eacute;tecte l&#39;activit&eacute; prot&eacute;ase limit&eacute;e aux cellules viables intactes. &Agrave; la mort de la cellule, le marqueur de prot&eacute;ase de cellules viables devienne inactif. Un rhodamine peptide imperm&eacute;able 110 (ou aminoluciferin) conjugu&eacute; substrat d&eacute;tecte l&#39;activit&eacute; de la prot&eacute;ase de cellules non viables qui ont perdu l&#39;int&eacute;grit&eacute; de la membrane. L&#39;essai multiplex peut d&eacute;tecter 200 cellules mortes dans une population de 10 000 cellules viables. Les r&eacute;actifs de substrat de prot&eacute;ase n&#39;endommagent pas les cellules viables au cours du test, donc la m&eacute;thode peut &ecirc;tre multiplex&eacute;e davantage avec d&#39;autres tests dans un format homog&egrave;ne. Ratiom&eacute;trique mesure des cellules viables et morts dans le m&ecirc;me &eacute;chantillon fournit un contr&ocirc;le interne qui peut &ecirc;tre utilis&eacute; pour normaliser les donn&eacute;es provenant d&#39;autres analyses cellulaires.

Un dosage homog&egrave;ne pour mesurer des cellules vivantes et mortes dans le m&ecirc;me &eacute;chantillon par d&eacute;tection des marqueurs de diff&eacute;rentes prot&eacute;ases.

Eine Methode, um gleichzeitig die relative Anzahl der lebenden und toten Zellen in Kultur bestimmen, durch die Einf&uuml;hrung einer Kombination von zwei Fluorogenic Substrate oder ein Fluorogenic und ein Luminogenic Protease-Substrat in der Probe wird beschrieben. Die Methode basiert auf der Erkennung von differenziellen allgegenw&auml;rtigen proteolytischen T&auml;tigkeiten im Zusammenhang mit intakten entwicklungsf&auml;higen Zellen und Zellen, die Membran Integrit&auml;t verloren haben. Eine Zelle-durchl&auml;ssigen Peptid-Aminofluorocoumarin-Substrat erkennt Protease-Aktivit&auml;t, die auf intakte entwicklungsf&auml;higen Zellen beschr&auml;nkt. Zelle Tod wird der lebensf&auml;higen Zellen Protease Marker inaktiv. Eine undurchl&auml;ssige Peptid Rhodamin 110 (oder Aminoluciferin) konjugierte Substrat erkennt Protease-Aktivit&auml;t aus nicht lebensf&auml;higes Zellen, die Membran Integrit&auml;t verloren haben. Der multiplex-Assay kann 200 Tote Zellen bei einer Gesamtbev&ouml;lkerung von 10.000 entwicklungsf&auml;higen Zellen erkennen. Die Protease-Substrat-Reagenzien im Laufe des Tests nicht lebensf&auml;hige Zellen Schaden anrichten, so kann die Methode Multiplexing werden weiter mit anderen Proben in einem einheitlichen Format. Ratiometrische Messung der lebensf&auml;higen und tote Zellen in derselben Probe stellt einen internen, der verwendet werden kann, um Daten aus anderen zellbasierte Assays zu normalisieren.

Eine homogene Assays lebenden und tote Zellen in derselben Probe zu messen, indem verschiedene Protease Marker.

Viene descritto un metodo per determinare contemporaneamente il relativo numero di cellule dal vivo e morte nella cultura con l&#39;introduzione di una combinazione di due substrati fluorogenic o un fluorogenic e un substrato della proteasi luminogenic nel campione. Il metodo si basa sulla rilevazione del differenziale onnipresente attivit&agrave; proteolitica associato intatte cellule vitali e le cellule che hanno perso l&#39;integrit&agrave; della membrana. Un substrato aminofluorocoumarin cellula permeabile peptide rileva attivit&agrave; proteasica limitato di cellule vitali intatte. Dopo la morte delle cellule, il marcatore di proteasi delle cellule vitali diventa inattivo. Un rodamina peptide impermeabile 110 (o aminoluciferin) substrato coniugato rileva l&#39;attivit&agrave; di proteasi dalle cellule non vitali che hanno perso l&#39;integrit&agrave; della membrana. Il test multiplex pu&ograve; rilevare 200 cellule morte in una popolazione di 10.000 cellule vitali. I reagenti di substrato proteasi non danneggiano cellule vitali nel corso del test, pertanto il metodo pu&ograve; essere multiplexato ulteriormente con altri dosaggi in un formato omogeneo. Raziometrici misura delle cellule vitali e morte nello stesso campione fornisce un controllo interno che pu&ograve; essere utilizzato per normalizzare i dati da altri cell-based assays.

Un dosaggio omogeneo per misurare le cellule dal vivo e morte nello stesso campione rilevando i marcatori di diverse proteasi.

&#x30B5;&#x30F3;&#x30D7;&#x30EB;&#x306B; 2 &#x3064;&#x306E;&#x86CD;&#x5149;&#x57FA;&#x677F;&#x3084;&#x3001;&#x86CD;&#x5149; luminogenic &#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30FC;&#x30BC;&#x57FA;&#x677F;&#x306E;&#x7D44;&#x307F;&#x5408;&#x308F;&#x305B;&#x3092;&#x5C0E;&#x5165;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x308A;&#x6587;&#x5316;&#x306E;&#x30E9;&#x30A4;&#x30D6;&#x3068;&#x30C7;&#x30C3;&#x30C9;&#x306E;&#x30BB;&#x30EB;&#x306E;&#x76F8;&#x5BFE;&#x6570;&#x3092;&#x540C;&#x6642;&#x306B;&#x6C7A;&#x5B9A;&#x3059;&#x308B;&#x65B9;&#x6CD5;&#x3092;&#x8AAC;&#x660E;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x30E1;&#x30BD;&#x30C3;&#x30C9;&#x306F;&#x305D;&#x306E;&#x307E;&#x307E;&#x5B9F;&#x884C;&#x53EF;&#x80FD;&#x306A;&#x30BB;&#x30EB;&#x304A;&#x3088;&#x3073;&#x819C;&#x306E;&#x5B8C;&#x5168;&#x6027;&#x3092;&#x5931;&#x3063;&#x3066;&#x3044;&#x308B;&#x30BB;&#x30EB;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x305F;&#x5DEE;&#x5206;&#x30E6;&#x30D3;&#x30AD;&#x30BF;&#x30B9;&#x86CB;&#x767D;&#x8CEA;&#x5206;&#x89E3;&#x6D3B;&#x6027;&#x306E;&#x691C;&#x51FA;&#x306B;&#x57FA;&#x3065;&#x3044;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x7D30;&#x80DE;&#x900F;&#x904E;&#x6027;&#x30DA;&#x30D7;&#x30C1;&#x30C9; aminofluorocoumarin &#x57FA;&#x677F;&#x3092;&#x305D;&#x306E;&#x307E;&#x307E;&#x5B9F;&#x884C;&#x53EF;&#x80FD;&#x306A;&#x30BB;&#x30EB;&#x3092;&#x5236;&#x9650;&#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30FC;&#x30BC;&#x6D3B;&#x6027;&#x3092;&#x691C;&#x51FA;&#x3057;&#x307E;&#x3059;&#x3002;&#x7D30;&#x80DE;&#x6B7B;&#x306B;&#x5B9F;&#x884C;&#x53EF;&#x80FD;&#x306A;&#x7D30;&#x80DE;&#x306E;&#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30FC;&#x30BC; &#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x306F;&#x975E;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306B;&#x306A;&#x308A;&#x307E;&#x3059;&#x3002;&#x30ED;&#x30FC;&#x30C0;&#x30DF;&#x30F3; 110 &#x306E;&#x4E0D;&#x6D78;&#x900F;&#x6027;&#x30DA;&#x30D7;&#x30C1;&#x30C9; (&#x307E;&#x305F;&#x306F; aminoluciferin) &#x5171;&#x5F79;&#x57FA;&#x677F;&#x819C;&#x306E;&#x5B8C;&#x5168;&#x6027;&#x3092;&#x5931;&#x3063;&#x3066;&#x3044;&#x308B;&#x8CA0;&#x3051;&#x306E;&#x7D30;&#x80DE;&#x304B;&#x3089;&#x306E;&#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30FC;&#x30BC;&#x6D3B;&#x6027;&#x3092;&#x691C;&#x51FA;&#x3057;&#x307E;&#x3059;&#x3002;&#x591A;&#x91CD;&#x30A2;&#x30C3;&#x30BB;&#x30A4; 200 &#x6B7B;&#x3093;&#x3060;&#x7D30;&#x80DE;&#x306B; 10,000 &#x306E;&#x5B9F;&#x884C;&#x53EF;&#x80FD;&#x306A;&#x30BB;&#x30EB;&#x306E;&#x4EBA;&#x53E3;&#x3092;&#x691C;&#x51FA;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;&#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30FC;&#x30BC;&#x306E;&#x57FA;&#x8CEA;&#x8A66;&#x85AC;&#x306F;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x306E;&#x30B3;&#x30FC;&#x30B9;&#x4E0A;&#x306E;&#x5B9F;&#x884C;&#x53EF;&#x80FD;&#x306A;&#x30BB;&#x30EB;&#x3092;&#x640D;&#x50B7;&#x3057;&#x306A;&#x3044;&#x3001;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x30E1;&#x30BD;&#x30C3;&#x30C9;&#x3092;&#x591A;&#x91CD;&#x5316;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3055;&#x3089;&#x306B;&#x3001;&#x540C;&#x4E00;&#x306E;&#x5F62;&#x5F0F;&#x306E;&#x4ED6;&#x306E;&#x8A66;&#x91D1;&#x3002;&#x30EC;&#x30B7;&#x30AA; &#x30E1;&#x30C8;&#x30EA;&#x30C3;&#x30AF;&#x6E2C;&#x5B9A;&#x540C;&#x3058;&#x30B5;&#x30F3;&#x30D7;&#x30EB;&#x306E;&#x5B9F;&#x884C;&#x53EF;&#x80FD;&#x306A;&#x6B7B;&#x3093;&#x3060;&#x7D30;&#x80DE;&#x306E;&#x4ED6;&#x306E;&#x7D30;&#x80DE;&#x306B;&#x57FA;&#x3065;&#x304F;&#x8A66;&#x91D1;&#x304B;&#x3089;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x3092;&#x6B63;&#x898F;&#x5316;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3001;&#x5185;&#x90E8;&#x306E;&#x30B3;&#x30F3;&#x30C8;&#x30ED;&#x30FC;&#x30EB;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;

&#x7570;&#x306A;&#x308B;&#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30FC;&#x30BC; &#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x3092;&#x691C;&#x51FA;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x308A;&#x3001;&#x540C;&#x3058;&#x30B5;&#x30F3;&#x30D7;&#x30EB;&#x306E;&#x30E9;&#x30A4;&#x30D6;&#x3068;&#x30C7;&#x30C3;&#x30C9; &#x30BB;&#x30EB;&#x3092;&#x6E2C;&#x5B9A;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x5747;&#x4E00;&#x306A;&#x30A2;&#x30C3;&#x30BB;&#x30A4;&#x3002;

&#xC0D8;&#xD50C;&#xC5D0; &#xB450; &#xAC1C;&#xC758; fluorogenic &#xAE30;&#xD310; &#xB610;&#xB294; fluorogenic &#xBC0F; luminogenic protease &#xAE30;&#xD310;&#xC758; &#xACB0;&#xD569;&#xC744; &#xB3C4;&#xC785; &#xD558; &#xC5EC; &#xB3D9;&#xC2DC;&#xC5D0; &#xBB38;&#xD654;&#xC5D0; &#xB77C;&#xC774;&#xBE0C; &#xD558; &#xACE0; &#xC8FD;&#xC740; &#xC138;&#xD3EC;&#xC758; &#xC0C1;&#xB300;&#xC801;&#xC778; &#xC22B;&#xC790;&#xB97C; &#xACB0;&#xC815; &#xD558;&#xB294; &#xBC29;&#xBC95;&#xC744; &#xC124;&#xBA85; &#xD569;&#xB2C8;&#xB2E4;. &#xBA54;&#xC11C;&#xB4DC;&#xAC00; &#xCC28;&#xB3D9; &#xC720;&#xBE44; &#xCFFC;&#xD130; &#xC2A4; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xD65C;&#xB3D9;&#xC744; &#xADF8;&#xB300;&#xB85C; &#xC2E4;&#xD589; &#xAC00;&#xB2A5;&#xD55C; &#xC140; &#xBC0F; &#xC140; &#xB9C9; &#xBB34;&#xACB0;&#xC131;&#xC744; &#xC783; &#xC5C8; &#xAD00;&#xB828; &#xB41C; &#xAC80;&#xC0C9;&#xC744; &#xAE30;&#xBC18;&#xC73C;&#xB85C; &#xD569;&#xB2C8;&#xB2E4;. &#xC138;&#xD3EC; &#xD22C;&#xACFC;&#xC131; &#xD3A9;&#xD0C0;&#xC774;&#xB4DC; aminofluorocoumarin &#xAE30;&#xD310; &#xADF8;&#xB300;&#xB85C; &#xAC00;&#xB2A5;&#xD55C; &#xC140;&#xC744; &#xC81C;&#xD55C; &#xD558;&#xB294; protease &#xD65C;&#xB3D9;&#xC744; &#xAC10;&#xC9C0;. &#xC138;&#xD3EC;&#xC758; &#xC8FD;&#xC74C;&#xC5D0; &#xB530;&#xB77C; &#xAC00;&#xB2A5;&#xD55C; &#xC138;&#xD3EC; &#xB2E8;&#xBC31;&#xC9C8; &#xB9C8;&#xCEE4; &#xBE44;&#xD65C;&#xC131;&#xD654; &#xB429;&#xB2C8;&#xB2E4;. &#xC2A4;&#xBA70;&#xB4E4; &#xC9C0; &#xC54A;&#xB294; &#xD3A9;&#xD0C0;&#xC774;&#xB4DC; rhodamine 110 (&#xB610;&#xB294; aminoluciferin) &#xD65C;&#xC6A9;&#xB41C; &#xAE30;&#xD310; &#xB9C9; &#xBB34;&#xACB0;&#xC131;&#xC744; &#xC783;&#xC5C8;&#xC744; &#xBBF8;&#xB2EC; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xB2E8;&#xBC31;&#xC9C8; &#xD65C;&#xB3D9;&#xC744; &#xAC10;&#xC9C0; &#xD569;&#xB2C8;&#xB2E4;. &#xBA40;&#xD2F0;&#xD50C;&#xB809;&#xC2A4; &#xBD84;&#xC11D; &#xACB0;&#xACFC; 10000 &#xAC00;&#xB2A5;&#xD55C; &#xC140;&#xC758; &#xC778;&#xAD6C;&#xC5D0;&#xC11C; 200 &#xC8FD;&#xC740; &#xC138;&#xD3EC;&#xB97C; &#xAC80;&#xCD9C;&#xD560; &#xC218; &#xC788;&#xB2E4;. Protease &#xAE30;&#xC9C8; &#xC2DC; &#xC57D; &#xBD84;&#xC11D; &#xACB0;&#xACFC;&#xC758; &#xACFC;&#xC815;&#xC744; &#xD1B5;&#xD574; &#xC2E4;&#xD589; &#xAC00;&#xB2A5;&#xD55C; &#xC138;&#xD3EC;&#xB97C; &#xC190;&#xC0C1; &#xB418;&#xC9C0; &#xC54A;&#xB3C4;&#xB85D;, &#xBA54;&#xC11C;&#xB4DC;&#xC758; &#xB2E4;&#xC911;&#xD654; &#xB530;&#xB77C;&#xC11C; &#xADE0;&#xC9C8; &#xD615;&#xD0DC;&#xB85C; &#xB2E4;&#xB978; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC744; &#xB354;. &#xB3D9;&#xC77C;&#xD55C; &#xC0D8;&#xD50C;&#xC5D0;&#xC11C; &#xC2E4;&#xC6A9;&#xC801;&#xC774; &#xACE0; &#xC8FD;&#xC740; &#xC138;&#xD3EC;&#xC758; &#xBE44;&#xC728; &#xCE21;&#xC815; &#xB2E4;&#xB978; &#xC138;&#xD3EC; &#xAE30;&#xBC18; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC5D0;&#xC11C; &#xB370;&#xC774;&#xD130; &#xD45C;&#xC900;&#xD654;&#xC5D0; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xB294; &#xB0B4;&#xBD80; &#xD1B5;&#xC81C;&#xB97C; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;.

&#xB2E4;&#xB978; &#xB2E8;&#xBC31;&#xC9C8; &#xB9C8;&#xCEE4;&#xB97C; &#xAC10;&#xC9C0; &#xD558; &#xC5EC; &#xB3D9;&#xC77C;&#xD55C; &#xC0D8;&#xD50C;&#xC5D0;&#xC11C; &#xB77C;&#xC774;&#xBE0C; &#xD558; &#xACE0; &#xC8FD;&#xC740; &#xC138;&#xD3EC;&#xB97C; &#xCE21;&#xC815; &#xD558;&#xB294; &#xC720;&#xD615;&#xC774; &#xAC19;&#xC740; &#xBD84;&#xC11D; &#xACB0;&#xACFC;.

Se describe un m&eacute;todo para determinar simult&aacute;neamente la cantidad relativa de c&eacute;lulas vivas y muertas en cultura introduciendo una combinaci&oacute;n de dos fluorogenic substratos o un fluorogenic y un sustrato de proteasa luminogenic en la muestra. El m&eacute;todo se basa en la detecci&oacute;n de las actividades proteol&iacute;ticas omnipresentes diferenciales asociados con c&eacute;lulas viables intactas y las c&eacute;lulas que han perdido la integridad de la membrana. Sustrato permeable a la c&eacute;lula p&eacute;ptido aminofluorocoumarin detecta actividad proteasa restringida a las c&eacute;lulas viables intactas. Sobre la muerte de la c&eacute;lula, el marcador de proteasa celular viable pasa a estar inactivo. Un rodamina p&eacute;ptido impermeable 110 (o aminoluciferin) substrato conjugado detecta actividad proteasa de c&eacute;lulas viables que han perdido la integridad de la membrana. El an&aacute;lisis m&uacute;ltiplex puede detectar 200 c&eacute;lulas muertas en una poblaci&oacute;n de 10.000 c&eacute;lulas viables. Los reactivos de sustrato de proteasa no da&ntilde;ar c&eacute;lulas viables en el transcurso del ensayo, por lo tanto el m&eacute;todo puede ser multiplexado con otros ensayos en un formato homog&eacute;neo. Medici&oacute;n radiom&eacute;trica de c&eacute;lulas viables y muertas en la misma muestra proporciona un control interno que puede utilizarse para normalizar los datos de otros ensayos celulares.

Un ensayo homog&eacute;neo para medir las c&eacute;lulas vivas y muertas en la misma muestra mediante la detecci&oacute;n de marcadores de proteasa diferentes.

Caspase activity assays in multi-well plate formats represent powerful tools for understanding experimental modulation of the apoptotic response. These assays are configured to exploit functional, biochemical, and temporal differences in substrate specificity and selectivity, which are useful in defining the magnitude and mechanism of a compound or treatment effect. New advances in fluorescent and luminescent chemistries now enable single addition "add-mix- measure" determinations of caspase activity directly in the sample plate with unprecedented sensitivity. Unlike other more cumbersome or laborious techniques, caspase activity induction or inhibition measures are quantifiable and definitive. The highlighted techniques in this chapter are cost efficient and allow for the rapid exploration of thousands of combinations and conditions.

Caspase activity assays.

Multiplexed assay chemistries provide for multiple measurements of cellular parameters within a single assay well. This experimental practice not only is more cost efficient but provides more informational content about a compound or treatment. For instance, multiplexed caspase activity assays can help establish the kinetics and magnitude of initiator and effector caspase induction by candidate compounds or treatments. The ability to combine the activity profiles within the same sample provides a level of normalization not possible with parallel assays. Furthermore, multiplexing caspase activity assays with viability and/or cytotoxicity assays can support conclusions regarding cytotoxic mechanism and provide normalization that may help correct for differences in cell number.

Multiplex caspase activity and cytotoxicity assays.

Bioluminescent assays couple a limiting component of a luciferase-catalyzed photon-emitting reaction to a variable parameter of interest, while holding the other components constant or non-limiting. In this way light output varies with the parameter of interest. This review describes three bioluminescent assay types that use firefly luciferase to measure properties of drugs and other xenobiotics which affect their absorption, distribution, metabolism, elimination and toxicity. First, levels of the luciferase enzyme itself are measured in gene reporter assays that place a luciferase cDNA under the control of regulatory sequences from ADMET-related genes. This approach identifies activators of nuclear receptors that regulate expression of genes encoding drug-metabolizing enzymes and drug transporters. Second, drug effects on enzyme activities are monitored with luminogenic probe substrates that are inactive derivatives of the luciferase substrate luciferin. The enzymes of interest convert the substrates to free luciferin, which is detected in a second reaction with luciferase. This approach is used with the drug-metabolizing CYP and monoamine oxidase enzymes, apoptosis-associated caspase proteases, a marker protease for non-viable cells and with glutathione-S-transferase to measure glutathione levels in cell lysates. Third, ATP concentration is monitored as a marker of cell viability or cell death and as a way of identifying substrates for the ATP-dependent drug transporter, P-glycoprotein. Luciferase activity is measured in the presence of a sample that supplies the requisite luciferase substrate, ATP, so that light output varies with ATP concentration. The bioluminescent ADMET assays are rapid and sensitive, amenable to automated high-throughput applications and offer significant advantages over alternative methods.

Bioluminescent assays for ADMET.

Measurement of cell membrane integrity has been widely used to assess chemical cytotoxity. Several assays are available for determining cell membrane integrity including differential labeling techniques using neutral red and trypan blue dyes or fluorescent compounds such as propidium iodide. Other common methods for assessing cytotoxicity are enzymatic "release" assays which measure the extra-cellular activities of lactate dehydrogenase (LDH), adenylate kinase (AK), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in culture medium. However, all these assays suffer from several practical limitations, including multiple reagent additions, scalability, low sensitivity, poor linearity, or requisite washes and medium exchanges. We have developed a new cytotoxicity assay which measures the activity of released intracellular proteases as a result of cell membrane impairment. It allows for a homogenous, one-step addition assay with a luminescent readout. We have optimized and miniaturized this assay into a 1536-well format, and validated it by screening a library of known compounds from the National Toxicology Program (NTP) using HEK 293 and human renal mesangial cells by quantitative high-throughput screening (qHTS). Several known and novel membrane disrupters were identified from the library, which indicates that the assay is robust and suitable for large scale library screening. This cytotoxicity assay, combined with the qHTS platform, allowed us to quickly and efficiently evaluate compound toxicities related to cell membrane integrity.

A bioluminescent cytotoxicity assay for assessment of membrane integrity using a proteolytic biomarker.

In vitro cytotoxicity testing has become an integral aspect of drug discovery because it is a convenient, costeffective, and predictive means of characterizing the toxic potential of new chemical entities. The early and routine implementation of this testing is testament to its prognostic importance for humans. Although a plethora of assay chemistries and methods exist for 96-well formats, few are practical and sufficiently sensitive enough for application in high throughput screening (HTS). Here we briefly describe a handful of the currently most robust and validated HTS assays for accurate and efficient assessment of cytotoxic risk. We also provide guidance for successful HTS implementation and discuss unique merits and detractions inherent in each method. Lastly, we discuss the advantages of combining specific HTS compatible assays into multi-parametric, same-well formats.

In vitro viability and cytotoxicity testing and same-well multi-parametric combinations for high throughput screening.

Testing the effects of compounds on the viability of cells grown in culture is widely used as a predictor of potential toxic effects in whole animals. Among the several alternative assays available, measuring the levels of ATP is the most sensitive, reliable, and convenient method for monitoring active cell metabolism. However, recently developed combinations of methods have made it possible to collect more information from in vitro cytotoxicity assays using standard fluorescence and luminescence plate readers. This chapter describes two assay methods. The first utilizes beetle luciferase for measuring the levels of ATP as a marker of viable cells. The second more recently developed multiplex method relies on selective measurement of three different protease activities as markers for viable, necrotic, and apoptotic cells. Data analysis from the measurement of three marker protease activities from the same sample provides a useful tool to help uncover the mechanism of cell death and can serve as an internal control to help identify assay artifacts.

Cytotoxicity testing: measuring viable cells, dead cells, and detecting mechanism of cell death.

Histone deacetylase (HDAC) enzymes modify the acetylation state of histones and other important proteins. Aberrant HDAC enzyme function has been implicated in many diseases, and the discovery and development of drugs targeting these enzymes is becoming increasingly important. In this article, the authors report the evaluation of homogeneous, single-addition, bioluminogenic HDAC enzyme activity assays that offer less assay interference by compounds in comparison to fluorescence-based formats. The authors assessed the key operational assay properties including sensitivity, scalability, reproducibility, signal stability, robustness (Z'), DMSO tolerance, and pharmacological response to standard inhibitors against HDAC-1, HDAC-3/NcoR2, HDAC-6, and SIRT-1 enzymes. These assays were successfully miniaturized to a 10 µL assay volume, and their suitability for high-throughput screening was tested in validation experiments using 640 drugs approved by the Food and Drug Administration and the Hypha Discovery MycoDiverse natural products library, which is a collection of 10 049 extracts and fractions from fermentations of higher fungi and contains compounds that are of low molecular weight and wide chemical diversity. Both of these screening campaigns confirmed that the bioluminogenic assay was high-throughput screening compatible and yielded acceptable performance in confirmation, counter, and compound/extract and fraction concentration-response assays.

A bioluminogenic HDAC activity assay: validation and screening.

Background: in vitro cytotoxicity testing provides a crucial means of ranking compounds for consideration in drug discovery. The choice of using a particular viability or cytotoxicity assay technology may be influenced by specific research goals. Objective: Although the high-throughput screening (HTS) utility is typically dependent upon sensitivity and scalability, it is also impacted by signal robustness and resiliency to assay interferences. Further consideration should be given to data quality, ease-of-use, reagent stability, and matters of cost-effectiveness. Methods: Here we focus on three main classes of assays that are at present the most popular, useful, and practical for HTS drug discovery efforts. These methods measure: i) viability by metabolism reductase activities; ii) viability by bioluminescent ATP assays; or iii) cytotoxicity by enzymes 'released' into culture medium. Multi-parametric technologies are also briefly discussed. Results/conclusion: Each of these methods has its relative merits and detractions; however multi-parametric methods using both viability and cytotoxicity markers may mitigate the inherent shortcomings of single parameter measures.

Update on in vitro cytotoxicity assays for drug development.

Drug-induced mitochondrial dysfunction is known to contribute to late stage compound attrition. Recently, assays that identify mitochondrial dysfunction have been developed but many require expensive reagents, specialized equipment, or specialized expertise such as isolation of mitochondria. Here, we validate a new 384-well format cell-based dual parameter assay that uses commonly available detection methods to measure both mitochondrial toxicity and cytotoxicity. In our initial evaluation, antimycin A, CCCP, nefazodone, flutamide, and digitonin were tested in K562 cells in both glucose- and galactose-supplemented media with a 2h incubation. The assay was able to correctly differentiate these compounds into mitochondrial toxicants and non-mitochondrial toxicants, and had excellent reproducibility. We next tested 74 compounds in K562 cells in both types of media and show that the assay was able to correctly identify some of the compounds as mitochondrial toxicants. Moreover, the assay could be simplified, without loss of information, by using K562 cells in galactose-containing medium alone. This simple, robust assay can be positioned as a rapid, early readout of mitochondrial and cellular toxicity. However, since the assay fails to identify some mitochondrial toxicants, further assays may be required to detect mitochondrial toxicity once lead compounds have been selected.

Andrew Niles

Promega Corporation

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology