This work was supported by grants from the Novartis Foundation for Medical-Biological Research and from the Research Pool of the University of Fribourg (to MW). We thank Li Zhao, Zhan Xu, and Solange Kharoubi Hess for technical support, as well as Radu Aricescu and Yvonne Jones (Oxford University, UK) for providing the pHLsec plasmid, and Thomas Sch&#252;rpf (Harvard Medical School) for helpful discussions.

1. Herstellung des Expressionsplasmids pHLsec-GZM <ol><li> Bereiten Gesamt-RNA aus menschlichen NK-Zellen (Primärzellen, wie in 26 oder der NK-Zelllinie NK-92 exprimieren, alle fünf menschlichen Gzms hergestellt) mit einer geeigneten RNA-Isolierungsverfahren und Umkehr transcribe unter Verwendung eines ersten cDNA synthetisieren Satz, nach dem manufacturer` s Empfehlungen. Verstärken GZM cDNA mittels PCR und Klon in pHLsec wie in 23 (Art Geschenk der Radu Aricescu und Yvonne Jones, University of Oxford, UK) beschrieben mit den AgeI und KpnI-Stellen (Abbildung 1). HINWEIS: Verwenden Sie die folgenden in Tabelle 1 angegebenen Primer. </li><li> Bestätigen richtigen Inserts durch Sequenzierung. Erweitern Sie die Expressionsplasmide in DH5a-Zellen und zu reinigen mit einem Endotoxin-freiem Plasmidisolierung Satz und befolgen Sie die Anweisungen des Herstellers. </li><li> Resuspendieren der gereinigten Plasmide in Endotoxin-freiem, sterilem Wasser bei einer Konzentration von 2 mg / ml und bei -20 ° C until Einsatz. </li></ol> 2. Expression von Gzms in HEK293T-Zellen <ol><li> Zubereitung von Reagenzien <ol><li> Bereiten Standardkulturmedium. In Dulbeccos Modified Eagle Medium (DMEM) mit hohem Glucosegehalt (25 mM), Glutamax (4 mM), Natriumpyruvat (1 mM) in 10% der Standard fötalem Kälberserum (FCS), Penicillin (100 Einheiten / ml), Streptomycin ( 100 ug / ml). </li><li> Bereiten Transfektionsmedium. Um Kulturmedium ohne Penicillin-Streptomycin hinzufügen 25 ug / ml Chloroquin (Add frisch am Tag der Transfektion von 1,000x Aktien in PBS, bei -20 ° C gelagert). </li><li> Vorbereitung serumfreien Kulturmedium: Zu einem serumfreien Medium für HEK293 Zellen hinzuzufügen Glutamin (4 mM), Penicillin (100 Einheiten / ml), Streptomycin (100 ug / ml) und 2,5 mg / l Amphotericin B. </li><li> Herzustellen, die in Tabelle 2 und der Filter mit einem 0,45 um Filter vor der Verwendung beschriebenen Lösungen: </li></ol></li><li> Ausbau HEK293T-Zellen <ol><li> Wachsen HEK293T-Zellen in 20 ml Kulture Medium unter Verwendung von 150 x 25 mm-Gewebekulturschalen. </li><li> Split-Zellen bei 80% Konfluenz (Split-Verhältnis von 1: 4, in der Regel jeden 3. Tag). Zellen lose befestigen, sie mechanisch ohne Trypsinierung abgelöst werden kann durch eine konsequente Auf- und Abpipettieren. </li><li> Plattenzellen in der Nacht vor der Transfektion auf 60-70% Konfluenz am Tag der Transfektion (Aussaatdichte ~ 2 x 10 5 Zellen / ml) zu ergeben. Eine übliche Vorbereitung Größe besteht aus 20 bis 25 Kulturschalen. </li></ol></li><li> Calciumphosphat-Transfektion <ol><li> 1 Stunde vor der Transfektion, ändern Sie die Standard-Kulturmedium auf 20 ml Transfektionsmedium. Kurz vor dem Ende dieser 1 h Inkubationsschritt mischen 400 ug Plasmid-DNA mit 10,95 ml ddH 2 0 und 1,55 ml 2 M CaCl 2 in 50-ml-Röhrchen (1 Röhrchen / 5 Gerichte). </li><li> 1 bis 2 min vor der Transfektion wurden anschließend 12,5 ml 2x HBS bis 12,5 ml der DNA-Calcium-Lösung, Mischung durch Umdrehen der Röhrchen und Inkubation für 30 s bei RT. </li><li> In the-Gemisch in 2.3.2 direkt auf die Zellen (5 ml pro Schale), tropfenweise in das Medium. Streuen gleichmäßig über die gesamte Fläche, Drehen des Mediums auf eine leicht orange Farbe. </li><li> Die Kulturschalen inkubieren 7-11 h in einem Brutschrank (37 ° C, 5% CO 2 in angefeuchteter Luft). Nach der Inkubation ist ein feiner Niederschlag sichtbar. Entfernen Sie das Transfektionsmedium gründlich vorsichtig mit vorgewärmten PBS und 20 ml Serum-freiem Kulturmedium in 2.1.3 vorbereitet. Inkubiere für 72 h in einem Brutschrank. </li><li> Analyse der Transfektion und Expressionseffizienz, indem eine Probe (~ 20 &amp; mgr; l) der Zellüberstand auf SDS-PAGE und Färbung mit Coomassie Brilliant Blue, ein Granzym Band visualisieren. </li></ol></li><li> Reinigung von Gzms aus Kulturüberstand von Nickel-IMAC <ol><li> Nach der Inkubation dekantieren die Zellkulturüberstände in 250-ml-Röhrchen und klar durch Zentrifugation. Führen Sie eine erste Spin (400 g, 10 min, 4 °C), die das Medium von abgelösten Zellen löschen wird. Den Überstand in frische 250 ml-Röhrchen und Schleudern bei 4000 xg, 30 min bei 4 ° C, um alle verbleibenden Zelltrümmer zu entfernen. </li><li> 5 ml 5 M NaCl, 6,25 ml 2 M Tris-Base (pH 8) und 1 ml von 0,25 M NiSO 4 pro 250 ml der geklärten Überstand. Den Überstand unter Verwendung eines 500 ml Vakuum-Filtereinheit (0,45 mm). </li><li> Äquilibrieren eine Nickel-IMAC-Säule mit His-Bindungspuffer A (10 Säulenvolumen, CV) mit einem geeigneten FPLC-System. </li><li> Gelten die geklärte Überstand auf die äquilibrierte Säule mit einer Fließgeschwindigkeit von 0,5 ml / min bei 4 ° C. </li><li> Nachdem der Überstand aufgebracht wurde, Waschsäule mit His-Bindungspuffer, bis die UV-Absorption Basislinie erreicht ist (normalerweise 10 CV). </li><li> Eluieren Gzms mit einem linearen 20 ml-Gradienten (0 bis 250 mM Imidazol) bei einer Flussrate von 0,5 ml / min, während das Sammeln 2 ml-Fraktionen. Analyse der Elutionsfraktionen durch SDS-PAGE und Coomassie-Färbung. </li></ol></li><li> Enterokinase (EK)Behandlung und Endreinigung durch Kationenaustauschchromatographie <ol><li> Pool GZM haltigen Fraktionen in einem Dialyseschlauch mit ≤10 MWCO. Bewahren Sie eine kleine Probe bei -20 ° C als Pre-EK Kontrolle. </li><li> In EK bei 0,02 Einheiten / 50 ml anfänglichen Überstand direkt auf die vereinigte Fraktion im Dialyseschlauch dialysiert und O / N (mindestens 16 h) bei RT in EK-Puffer (4 L, ändern Puffer einmal). </li><li> Am nächsten Tag analysiert EK behandelt Gzms im Vergleich zur vorge EK Kontrolle durch SDS-PAGE und Coomassie-Färbung. </li><li> Wenn N-terminale Verarbeitung abgeschlossen ist, ändern Dialysepuffer auf S Puffer A (4 l) dialysiert und für weitere 4 h bei RT. Filter Dialysat (0,45 um). </li><li> Äquilibrieren eine S-S-Säule mit Puffer A. Laden der Probe auf die Säule mit einer Fließgeschwindigkeit von 0,5 ml / min bei 4 ° C. </li><li> Nach der Probenbeladung, waschen Sie die Säule mit S-Puffer A, bis die UV-Absorption Basislinie erreicht ist (ca. 10 CV). Eluieren die Gzms mit einer 30 ml linearen Gradienten (150 bis 1000 mM NaCl). GZMA eLauten bei ~ 650 mM NaCl, GzmB bei ~ 700 mM NaCl und GzmM bei ~ 750 mM NaCl. </li><li> Analysieren Elutionsfraktionen durch SDS-PAGE und kolorimetrische Assays (siehe unten). Pool enthaltenden Fraktionen Gzms und Konzentrat (etwa 30-fach, auf eine Konzentration von etwa 100 uM) in Spinfilter (15 ml, 10 MWCO). Aliquot der konzentrierten GZM Vorbereitungen und bei -80 ° C. </li></ol></li></ol> 3. Prüfung GZM Aktivität <ol><li> Zubereitung von Reagenzien <ol><li> Vorbereitung kolorimetrischen Assay-Puffer: 50 mM Tris-Base, pH 7. <ol><li> Für die Messung der GZMA fügen Na-CBZ-L-Lysin thiobenzyl ester (S-Bzl) (BLT) mit dem Assay-Puffer auf eine Endkonzentration von 0,2 mM und 5,5&#39;-Dithio-bis (2-nitrobenzoesäure) (DTNB) in einer Konzentration von 0,22 mM. Für GzmB umfassen 0,2 mM Boc-Ala-Ala-Asp-S-Bzl (AAD) und 0,22 mM DTNB. Für GzmM umfassen 0,2 mM Suc-Ala-Ala-Pro-Leu-p-Nitroanilid (pNA) (AAPL). Synthetische Peptide werden von entsprechenden Stammlösungen in DMSO (, stored bei -20 ° C) zu dem Puffer frisch vor dem Assay. </li></ol></li><li> Vorbereitung Spaltungstestpuffer: 100 mM NaCl, 50 mM Tris-Base, pH 7,5 </li></ol></li><li> Kolorimetrische Assays <ol><li> Verteilen Sie kleine GZM Proben (&lt;5 &amp; mgr; l) von Elutionsfraktionen oder konzentrierte Aktien in den 96-Loch-Flachbodenplatten. Fügen Sie eine positive Kontrolle (getestet GZM Zubereitungen oder rohe NK Zell-Lysate) und eine Negativkontrolle (nur Puffer). </li><li> Füge 100 &amp; mgr; l Assay-Puffer in jede Vertiefung (unter Verwendung einer Mehrkanalpipette). Inkubieren für 10 min bei 37 ° C. Messen der OD bei 405 nm in einem Mikroplatten-Lesegerät. </li></ol></li><li> Spaltungsassay <ol><li> Für Spaltungs-Assays unter Verwendung von gereinigten Substrate, Co-Inkubation ein Protein-Substrat (native oder rekombinante, 100-400 nM; einige Beispiele für effiziente GZM Substrate werden in der Ergebnisbereich und in dem Hinweis am Ende dieses Abschnitts dargestellt) mit seriellen Verdünnungen einer GZM (beginnend bei 400 nM) in Assay-Puffer für verschiedene Zeiten.Analyse durch SDS-PAGE und Silberfärbung oder Coomassie; oder durch Western-Blot unter Verwendung spezifischer Antikörper. </li><li> Zur Abspaltung Assays unter Verwendung Zelllysaten auszusetzen 10 6 HeLa-Zellen (oder einen beliebigen verfügbaren Tumorzelllinie) in 1 ml Testpuffer und Lyse durch Einfrieren in einem Ethanol / Trockeneis-Bad und Auftauen bei 37 ° C dreimal. Entfernen von Zelltrümmern durch Zentrifugation (15.000 × g für 10 min bei 4 ° C). </li><li> Co-Inkubation mit dem geklärten Lysat Gzms in verschiedenen Konzentrationen und Zeiten wie oben. Spaltung wird durch Immunoblotting unter Verwendung von geeigneten Antikörpern nachgewiesen. HINWEIS: Einige Beispiele für Bona-fide-Substrate, für die kommerzielle Antikörper verfügbar sind: Bid (BH3-Domäne interagieren Tod Agonist) und Caspase 3 durch GzmB 27,28 gespalten; mehrere heterogene Kern Ribonukleoproteine ​​(hnRNPA1, A2 und U) durch GZMA 29 gespalten wird; Cytomegalovirus Phosphoprotein 71 durch GzmM 30. </li></ol></li></ol>

<ol>
	<li>Chowdhury, D., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Death+by+a+thousand+cuts:+granzyme+pathways+of+programmed+cell+death.">Death by a thousand cuts: granzyme pathways of programmed cell death.</a> Annu Rev Immunol. 26, 389-420 (2008).</li><li>Thiery, J., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perforin:+a+key+pore-forming+protein+for+immune+control+of+viruses+and+cancer.">Perforin: a key pore-forming protein for immune control of viruses and cancer.</a> Subcell Biochem. 80, 197-220 (2014).</li><li>Krensky, A. M., Clayberger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+clinical+relevance+of+granulysin.">Biology and clinical relevance of granulysin.</a> Tissue Antigens. 73, 193-198 (2009).</li><li>Bovenschen, N., Kummer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orphan+granzymes+find+a+home.">Orphan granzymes find a home.</a> Immunol Rev. 235, 117-127 (2010).</li><li>Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granzyme+A+activates+another+way+to+die.">Granzyme A activates another way to die.</a> Immunol Rev. 235, 93-104 (2010).</li><li>Ewen, C. L., Kane, K. P., Bleackley, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quarter+century+of+granzymes.">A quarter century of granzymes.</a> Cell death and differentiation. 19, 28-35 (2012).</li><li>Hiebert, P. R., Granville, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granzyme+B+in+injury,+inflammation,+and+repair.">Granzyme B in injury, inflammation, and repair.</a> Trends Mol Med. 18, 732-741 (2012).</li><li>Afonina, I. S., Cullen, S. P., Martin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+and+non-cytotoxic+roles+of+the+CTL/NK+protease+granzyme.">Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme.</a> B. Immunol Rev. 235, 105-116 (2010).</li><li>Walch, M., 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=Cytotoxic+cells+kill+intracellular+bacteria+through+granulysin-mediated+delivery+of+granzymes.">Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes.</a> Cell. 157, 1309-1323 (2014).</li><li>Lee, W. Y., 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=Invariant+natural+killer+T+cells+act+as+an+extravascular+cytotoxic+barrier+for+joint-invading+Lyme+Borrelia.">Invariant natural killer T cells act as an extravascular cytotoxic barrier for joint-invading Lyme Borrelia.</a> Proc Natl Acad Sci U S A. , (2014).</li><li>Kapelski, S., de Almeida, M., Fischer, R., Barth, S., Fendel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimalarial+activity+of+granzyme+B+and+its+targeted+delivery+by+a+granzyme+B-scFv+fusion+protein.">Antimalarial activity of granzyme B and its targeted delivery by a granzyme B-scFv fusion protein.</a> Antimicrob Agents Chemother. , (2014).</li><li>Thiery, J., Walch, M., Jensen, D. K., Martinvalet, D., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+cytotoxic+T+cell+and+NK+granules+and+purification+of+their+effector+proteins.">Isolation of cytotoxic T cell and NK granules and purification of their effector proteins.</a> Curr Protoc Cell Biol. Chapter 3 (Unit3 37), (2010).</li><li>Shi, L., Yang, X., Froelich, C. J., Greenberg, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+use+of+granzyme+B.">Purification and use of granzyme B.</a> Methods Enzymol. 322, 125-143 (2000).</li><li>Masson, D., Tschopp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+family+of+serine+esterases+in+lytic+granules+of+cytolytic+T+lymphocytes.">A family of serine esterases in lytic granules of cytolytic T lymphocytes.</a> Cell. 49, 679-685 (1987).</li><li>Lorentsen, R. H., Fynbo, C. H., Thogersen, H. C., Etzerodt, M., Holtet, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression,+refolding,+and+purification+of+recombinant+human+granzyme+B.">Expression, refolding, and purification of recombinant human granzyme B.</a> Protein Expr Purif. 39, 18-26 (2005).</li><li>Sun, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+purification+of+recombinant+human+granzyme+B+from+Pichia+pastoris.">Expression and purification of recombinant human granzyme B from Pichia pastoris.</a> Biochem Biophys Res Commun. 261, 251-255 (1999).</li><li>Xia, Z., 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=Expression+and+purification+of+enzymatically+active+recombinant+granzyme+B+in+a+baculovirus+system.">Expression and purification of enzymatically active recombinant granzyme B in a baculovirus system.</a> Biochem Biophys Res Commun. 243, 384-389 (1998).</li><li>Stahnke, B., 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=Granzyme+B-H22(scFv),+a+human+immunotoxin+targeting+CD64+in+acute+myeloid+leukemia+of+monocytic+subtypes.">Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes.</a> Mol Cancer Ther. 7, 2924-2932 (2008).</li><li>Gehrmann, M., 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=A+novel+expression+and+purification+system+for+the+production+of+enzymatic+and+biologically+active+human+granzyme.">A novel expression and purification system for the production of enzymatic and biologically active human granzyme.</a> B. J Immunol Methods. 371, 8-17 (2011).</li><li>Metkar, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+cell+granule-mediated+apoptosis:+perforin+delivers+granzyme+B-serglycin+complexes+into+target+cells+without+plasma+membrane+pore+formation.">Cytotoxic cell granule-mediated apoptosis: perforin delivers granzyme B-serglycin complexes into target cells without plasma membrane pore formation.</a> Immunity. 16, 417-428 (2002).</li><li>Motyka, B., 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=Mannose+6-phosphate/insulin-like+growth+factor+II+receptor+is+a+death+receptor+for+granzyme+B+during+cytotoxic+T+cell-induced+apoptosis.">Mannose 6-phosphate/insulin-like growth factor II receptor is a death receptor for granzyme B during cytotoxic T cell-induced apoptosis.</a> Cell. 103, 491-500 (2000).</li><li>Pinkoski, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+and+trafficking+of+granzyme+B+in+target+cells+during+granzyme+B-perforin-mediated+apoptosis.">Entry and trafficking of granzyme B in target cells during granzyme B-perforin-mediated apoptosis.</a> Blood. 92, 1044-1054 (1998).</li><li>Aricescu, A. R., Lu, W., Jones, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time-+and+cost-efficient+system+for+high-level+protein+production+in+mammalian+cells.">A time- and cost-efficient system for high-level protein production in mammalian cells.</a> Acta Crystallogr D Biol Crystallogr. 62, 1243-1250 (2006).</li><li>Fukuchi, K., 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=Activity+assays+of+nine+heterogeneous+promoters+in+neural+and+other+cultured+cells.">Activity assays of nine heterogeneous promoters in neural and other cultured cells.</a> In Vitro Cell Dev Biol Anim. 30 A, 300-305 (1994).</li><li>Tran, T. V., Ellis, K. A., Kam, C. M., Hudig, D., Powers, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dipeptidyl+peptidase+I:+importance+of+progranzyme+activation+sequences,+other+dipeptide+sequences,+and+the+N-terminal+amino+group+of+synthetic+substrates+for+enzyme+activity.">Dipeptidyl peptidase I: importance of progranzyme activation sequences, other dipeptide sequences, and the N-terminal amino group of synthetic substrates for enzyme activity.</a> Arch Biochem Biophys. 403, 160-170 (2002).</li><li>Somanchi, S. S., Senyukov, V. V., Denman, C. J., Lee, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion,+purification,+and+functional+assessment+of+human+peripheral+blood+NK+cells.">Expansion, purification, and functional assessment of human peripheral blood NK cells.</a> Journal of visualized experiments : JoVE. , (2011).</li><li>Barry, M., 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=Granzyme+B+short-circuits+the+need+for+caspase+8+activity+during+granule-mediated+cytotoxic+T-lymphocyte+killing+by+directly+cleaving+Bid.">Granzyme B short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving Bid.</a> Mol Cell Biol. 20, 3781-3794 (2000).</li><li>Darmon, A. J., Nicholson, D. W., Bleackley, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+apoptotic+protease+CPP32+by+cytotoxic+T-cell-derived+granzyme+B.+.">Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B. .</a> Nature. 377, 446-448 (1995).</li><li>Rajani, D. K., Walch, M., Martinvalet, D., Thomas, M. P., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+RNA+processing+during+immune-mediated+programmed+cell+death.">Alterations in RNA processing during immune-mediated programmed cell death.</a> Proc Natl Acad Sci U S A. 109, 8688-8693 (2012).</li><li>Domselaar, R., 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=Noncytotoxic+inhibition+of+cytomegalovirus+replication+through+NK+cell+protease+granzyme+M-mediated+cleavage+of+viral+phosphoprotein+71.">Noncytotoxic inhibition of cytomegalovirus replication through NK cell protease granzyme M-mediated cleavage of viral phosphoprotein 71.</a> J Immunol. 185, 7605-7613 (2010).</li><li>Thiery, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perforin+activates+clathrin-+and+dynamin-dependent+endocytosis,+which+is+required+for+plasma+membrane+repair+and+delivery+of+granzyme+B+for+granzyme-mediated+apoptosis.">Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis.</a> Blood. 115, 1582-1593 (2010).</li><li>Thiery, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perforin+pores+in+the+endosomal+membrane+trigger+the+release+of+endocytosed+granzyme+B+into+the+cytosol+of+target+cells.">Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells.</a> Nat Immunol. 12, 770-777 (2011).</li><li>Thomas, 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=Leukocyte+protease+binding+to+nucleic+acids+promotes+nuclear+localization+and+cleavage+of+nucleic+acid+binding+proteins.">Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.</a> J Immunol. 192, 5390-5397 (2014).</li><li>Jacquemin, G., 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=Granzyme+B-induced+mitochondrial+ROS+are+required+for+apoptosis.">Granzyme B-induced mitochondrial ROS are required for apoptosis.</a> Cell death and differentiation. , (2014).</li><li>Kummer, J. A., Kamp, A. M., Citarella, F., Horrevoets, A. J., Hack, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+human+recombinant+granzyme+A+zymogen+and+its+activation+by+the+cysteine+proteinase+cathepsin+C.">Expression of human recombinant granzyme A zymogen and its activation by the cysteine proteinase cathepsin C.</a> The Journal of biological chemistry. 271, 9281-9286 (1996).</li><li>Hagn, M., Sutton, V. R., Trapani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+colorimetric+assay+that+specifically+measures+Granzyme+B+proteolytic+activity:+hydrolysis+of+Boc-Ala-Ala-Asp-S-Bzl.">A colorimetric assay that specifically measures Granzyme B proteolytic activity: hydrolysis of Boc-Ala-Ala-Asp-S-Bzl.</a> Journal of visualized experiments : JoVE. , e52419 (2014).</li><li>Huang, M. T., Gorman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervening+sequences+increase+efficiency+of+RNA+3'+processing+and+accumulation+of+cytoplasmic+RNA.">Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA.</a> Nucleic Acids Res. 18, 937-947 (1990).</li><li>Luthman, H., Magnusson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+polyoma+DNA+transfection+of+chloroquine+treated+cells.">High efficiency polyoma DNA transfection of chloroquine treated cells.</a> Nucleic Acids Res. 11, 1295-1308 (1983).</li><li>Magee, A. I., Grant, D. A., Hermon-Taylor, J., Offord, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+one-stage+method+for+assay+of+enterokinase+activity+by+release+of+radiolabelled+activation+peptides+from+alpha-N-[3H]acetyl-trypsinogen+and+the+effect+of+calcium+ions+on+the+enzyme+activity.">Specific one-stage method for assay of enterokinase activity by release of radiolabelled activation peptides from alpha-N-[3H]acetyl-trypsinogen and the effect of calcium ions on the enzyme activity.</a> Biochem J. 197, 239-244 (1981).</li></ol>

The authors have nothing to disclose.

Die klassische und ausgiebig untersucht Rolle GZMA und GzmB ist die Induktion von Apoptose in Säugetierzellen nach ihrer zytosolischen Lieferung durch den porenbildendes Protein PFN 1. Kürzlich wurde die zytotoxische Spektrum der Gzms signifikant von Säugetierzellen, Bakterien 9,10 sowie bestimmte Parasiten 11 verbreitert. Darüber hinaus sind die nicht-klassischen, extrazelluläre Funktionen GZMA und GzmB sowie die biologische Bedeutung der verschiedenen Waisen Gzms noch unklar. Dahe...

Die Gzms sind eine Familie von hoch homologen Serinproteasen in spezialisierten Lysosomen von CTL und NK-Zellen 1 lokalisiert. Die zytotoxischen Granula dieser Killerzellen enthalten auch die Membran-störenden Proteinen PFN und GNLY, die gleichzeitig mit den Gzms bei Erkennung einer Zielzelle für die Eliminierung 2,3 bestimmt werden freigegeben. Es gibt fünf Gzms beim Menschen (GZMA, B, H, K und M) und 10 Gzms in Mäusen (GZMA - G, K, M und N). GZMA und GzmB sind die häufigsten und umfassend in Menschen und Mäusen 1 untersucht. Allerdings haben neuere Studien begonnen, die Zelltod Wege sowie die zusätzlichen biologischen Wirkungen von den anderen, so genannten Orphan-Gzms in Gesundheit und Krankheit 4 vermittelt zu untersuchen. Das am besten bekannte Funktion der Gzms, insbesondere GZMA und GzmB, ist die Induktion von programmiertem Zelltod in Säugerzellen, wenn sie von PFN 5,6 in die Zielzellen geliefert. JedochZeigten neuere Studien auch extrazelluläre Effekte der Gzms mit tiefgreifenden Auswirkungen auf die Immunregulation und Entzündungen, unabhängig von zytosolischen Lieferung durch PFN 7,8. Das Spektrum von Zellen, die effizient nach cytosolischen Eingabe der Gzms getötet wurde vor kurzem auch aus Säugerzellen, Bakterien 9,10 und sogar bestimmte Parasiten 11 verbreitert. Diese jüngsten Entdeckungen auf ein ganz neues Feld für GZM Forscher eröffnet. Daher wird eine kostengünstige, High-Yield-Säugetier-Expressionssystem deutlich den Weg für die künftige Studien erleichtern. Nativen menschlichen, Maus und Ratte Gzms wurden erfolgreich aus der Kornfraktion von CTL und NK-Zellen Linien 12-14 gereinigt. Jedoch in unseren Händen die Ausbeute solcher Reinigungsverfahren ist in dem Bereich von weniger als 0,1 mg / l Zellkultur (unveröffentlichte Beobachtung und 12). Darüber hinaus die chromatographische Auflösung eines einzelnen GZM ohne Verunreinigung durch other Gzms und / oder Proteine, die ebenfalls vorhanden sind, in den Granulaten ist eine Herausforderung (unveröffentlichte Daten und 12,14). Rekombinante Gzms wurden Bakterien 15, Hefe 16, Insektenzellen 17 und auch in Säugerzellen, wie HEK 293 18,19 hergestellt. Nur die Säugetier-Expressionssysteme tragen das Potenzial, rekombinanten Enzyme mit posttranslationalen Modifikationen identisch mit dem nativen zytotoxischen Protein zu produzieren. Posttranslationale Modifikationen der spezifischen Aufnahme durch Endozytose und die intrazelluläre Lokalisation der Protease in Zielzellen 20-22 gebracht. Daher kann unter Verwendung pHLsec 23 (ein freundliches Geschenk von Radu Aricescu und Yvonne Jones, University of Oxford, UK) als Plasmidrückgrat für GZM Ausdruck haben wir ein einfaches, zeit- und kosteneffizientes System für High-Yield-Proteinproduktion in HEK293T-Zellen. pHLsec kombiniert eine CMV-Enhancer mit einem Huhn Β-Actin-Promotor; Zusammen bilden diese Elemente Demonstrationented der stärkste Promotor-Aktivität in verschiedenen Zelllinien 24. Zusätzlich enthält das Plasmid ein Kaninchen Β-Globin-Intron, optimierte Kozak und Sekretionssignalen, einen Lys-6xHis-Tag und einem Poly-A-Signal. Inserts können bequem zwischen dem Sekretionssignal und dem Lys-6xHis-tag (Abbildung 1) eine optimale Expression und Sekretion Effizienz für Proteine ​​fehlen geeignete N-terminalen Domänen geklont werden. Für die Expression der Gzms, die endogene Sekretionssignal-Sequenz mit dem Sekretionssignal von dem Vektor, gefolgt von einer Enterokinase (EK) Website (DDDDK) versehen ist, so dass wir ersetzt EK Behandlung aktiviert die sezernierten Gzms (aktiv Gzms beginnen mit dem N-terminalen Aminosäuresequenz IIGG 25). Zusätzlich zugunsten für dieses Verfahren, HEK293T Zellen schnell wachsen in günstiges Medium, wie Dulbeccos modifiziertes Eagle-Medium (DMEM) und sind für eine kosteneffiziente Calciumphosphat-Transfektionsverfahren gut geeignet.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td>Total RNA isolation kit</td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase</td>
			<td>Invitrogen</td>
			<td>18064-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td>M0530L</td>
			<td></td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>EX-CELL 293 Serum-Free Medium for HEK 293 Cells</td>
			<td>Sigma</td>
			<td>14571C</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td></td>
		</tr>
		<tr>
			<td>SnakeSkin Dialysis Tubing, 10K MWCO</td>
			<td>Thermo Scientific</td>
			<td>68100</td>
			<td></td>
		</tr>
		<tr>
			<td>Enterokinase from bovine intestine</td>
			<td>Sigma</td>
			<td>E4906</td>
			<td>recombinant, &#8805;20&#160;units/mg protein</td>
		</tr>
		<tr>
			<td>HisTrap Excel 5ml-column</td>
			<td>GE Healthcare</td>
			<td>17-3712-06&#160;</td>
			<td>Nickel IMAC</td>
		</tr>
		<tr>
			<td>HiTrap SP HP 5 ml-column</td>
			<td>GE Healthcare</td>
			<td>17-1152-01&#160;</td>
			<td>S column</td>
		</tr>
		<tr>
			<td>N-&#945;-Cbz-L-lysine thiobenzylester (BLT)</td>
			<td>Sigma</td>
			<td>C3647</td>
			<td>GzmA substrate</td>
		</tr>
		<tr>
			<td>Boc-Ala-Ala-Asp-S-Bzl (AAD)</td>
			<td>MP Biomedicals</td>
			<td>2193608 _10mg</td>
			<td>GzmB substrate</td>
		</tr>
		<tr>
			<td>Suc-Ala-Ala-Pro-Leu-p-nitroanilide (AAPL)</td>
			<td>Bachem</td>
			<td></td>
			<td>GzmM substrate</td>
		</tr>
		<tr>
			<td>5,5&#8242;-dithio-bis(2-nitrobenzoic acid) (DTNB)</td>
			<td>Sigma</td>
			<td>D8130</td>
			<td>Ellman`s reagent</td>
		</tr>
	</tbody>
</table>

a high yield and cost-efficient expression system of human granzymes in mammalian cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

Im folgenden Abschnitt werden wir eine vollständige Dokumentation eines GZMA Vorbereitung zu präsentieren, um die Methode zu beleuchten. Wir haben auch erfolgreich gereinigt GzmB und GzmM, die ähnliche Ergebnisse in Bezug auf Reinigungsleistung und Aktivität. Doch von diesen später Vorbereitungen werden wir nur zeigen einige ausgewählte Stücke von Daten. Wurden GzmB Präparate aus 293T-Zellen nach dem aktuellen Protokoll in mehreren veröffentlichten Studien verwendet, Hervorhebung ihrer Tätigkeit in verschieden...

Watch this Scientific Journal Video about A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells at JoVE.com

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

Eine hohe Ausbeute und kosteneffiziente Expression System der Menschen Granzyme in Säugerzellen

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

a-high-yield-cost-efficient-expression-system-human-granzymes

Research

JoVE Journal

Biology

9.7K Aufrufe.  Boston Children’s Hospital and Harvard Medical School. We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease. 

Serie: A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Mit dem Gene Pulser MXcell Elektroporation System zur Primary Cells mit hohem Wirkungsgrad zu transfizieren

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Forschung

Biologie

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Verwendung eines automatisierten Cell Counter zu Genexpressionsstudien Simplify: siRNA Knockdown von IL-4 abhängige Genexpression in Namalwalzellen

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

Chromatin Immunopräzipitation (ChIP) zur Assay Dynamische Histonmodifikationen in Activated Gene Expression in menschlichen Zellen

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagenese und funktionale Analyse von Ionenkanälen heterolog in Säugerzellen exprimierten

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Absolute Quantenausbeute Messung von Pulverproben

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Effiziente Ableitung von Human Cardiac Vorstufen und Kardiomyozyten aus pluripotenten humanen embryonalen Stammzellen mit Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Analyse des Zellzyklus Position in Säugerzellen

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

Expressionsanalyse von Säugetier-Linker-Histon-Subtypen

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

Visualisierung von endoplasmatischen Retikulum lokalisierte mRNAs in Säugerzellen

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA Screening zu Ubiquitin und Ubiquitin-ähnlichen System Regler der biologischen Stoffwechselwege in kultivierten Säugerzellen identifizieren

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...