Diese Arbeit wurde vom National Institutes of Health (CA142664) unterstützt.

1. Detektion von ROS mit Carboxy-H 2 DCFDA Carboxy-H 2 DCFDA ist nicht fluoreszierenden, aber in Anwesenheit von ROS, wenn dieses Reagenz oxidiert wird, wird es grün fluoreszierend. <ol><li> Unmittelbar vor der Verwendung, eine frische Stammlösung von Carboxy-H 2 DCFDA in sterile Dimethylsulfoxid (DMSO) oder 100% Ethanol. Vermeiden Sie mehrere Tau-/ freeze-Zyklen Ihrer Farbstoff. </li><li> Waschen Sie die Zellen mit HEPES gepufferte Salzlösung (HBSS) oder Phosphat-gepufferter Salzlösung (PBS), um Spuren der ursprünglichen Medium zu entfernen. </li><li> Laden Sie die Zellen mit dem Farbstoff (und die Kontrolle Farbstoff, vgl. Anm. Abschnitt). In diesem Test verwendeten wir Jurkat, einer menschlichen Leukämie-Zelllinie. Verwenden Sie Carboxy-H 2 DCFDA in einer Endkonzentration von 1 &amp;mgr; in regelmäßigen Kulturmedium mit reduzierter Serum (2%). </li><li> Inkubieren Sie die Kulturen für 30 Minuten im Dunkeln, in einer konventionellen Inkubator (37 ° C, 5% CO2). Entsorgen Sie alle unbenutzten Farbstofflösungen. </li><li> Entfernen carboxy-H 2 DCFDA Medium und waschen zweimal mit HBSS oder PBS. Von diesem Schritt, schützen Sie Ihre Zellen aus Licht. </li><li> Fügen Sie frisches Medium mit Ihrem Medikament der Wahl zum Carboxy-H 2 DCFDA-beladenen Zellen und inkubieren, wie gewünscht. Für dieses Beispiel verwenden wir H 2 O 2 (0,03%) für 1 Stunde. </li><li> Bewerten ROS durch eine sofortige Analyse Ihrer Zellen mittels Durchflusszytometrie mit dem FL1-Kanal (grüne Fluoreszenz), oder durch Fluoreszenz-Reader oder durch Fluoreszenz-Mikroskopie. Die Fluoreszenz kann durch Anregungs-und Emissionswellenlänge, die sich für grüne Fluoreszenz nachgewiesen werden. </li></ol> Hinweis: Die Kontrollen sollten Carboxy-H 2 DCFDA-geladen unbehandelten Zellen und ungefärbten unbehandelten Zellen beinhalten. Carboxy-H 2 DCFDA ist bekannt, Peroxide erkennen, sondern könnten auch durch andere ROS oxidiert werden. Dieses Reagenz kann auch durch andere Mittel geändert werden, Oxidation unempfindlich Kontrolle Farbstoff wie 5 - (und-6)-Carboxy-2 &#39;, 7&#39;-Dichlorofluorescein Diacetat (Carboxy-DCFDA) sollte daher in den Test einbezogen werden. 2. Messung von Stickstoffmonoxid (NO)-Produktion Sie müssen Sulfanilamid und N-1-napthylethylenediamine dihydrochlorid (NED)-Lösungen, und Nitrit-Standard. Dieser Test wird als Griess-Assay. NED: Mach eine 0,1% ige Lösung von N-1-napthylethylenediamine dihydrochlorid in sterile water.Sulfanilamide verdünnt: Machen Sie eine 1% ige Lösung von Sulfanilamid in 5%-iger Phosphorsäure verdünnt. Nitrit Standard: Verdünnen Sie die 0,1 Standard-Lager Natriumnitrit zu 100 &amp;mgr; in steriles Medium, keine serielle Verdünnung in dem gleichen Medium. Lagerbedingungen: Chemikalien, die als vom Hersteller bei Raumtemperatur geleitet. Nach dem Auflösen sind NED und Sulfanilamide Lösungen sofort nach Gebrauch bei 4 ° C gelagert, in der Dunkelheit, und für maximal 3 Monate. <ol><li> Kultur-Zellen in einer 96-Well-Platte,Verwendung dreifach für jede Bedingung und umfassen angemessene Kontrollen nach Ihren Experimenten. </li><li> Gönnen Zellen die NO-Produktion zu induzieren. In unserem Experiment nutzen wir Lipopolysaccharid (LPS) (100 ng / ml) und rekombinantem IL-4, um unsere Zellen zu behandeln. In diesem Protokoll verwendeten wir RAW 264.7, eine Maus-Makrophagen-Zelllinie (Maus leukämischen Monozyten-Makrophagen-Zelllinie). </li><li> Am Tag des Tests, bringen beide Reagenzien auf Raumtemperatur. </li><li> Spin Ihrem Teller, sammeln Zellüberständen und Transfer 50 ul, um eine neue 96-Well-Platte. Bereiten Grenzverdünnung Brunnen Ihrer Standard-Stammlösung zu einer Standard-Kurve machen. </li><li> Add 50 ul Sulfanilamide Lösung für jede Probe und Kontrolle gut und gut mischen. </li><li> Inkubieren bei Raumtemperatur für 10 Minuten im Dunkeln. </li><li> Add 50 ul N-1-napthylethylenediamine dihydrochlorid Lösung für jede Probe und Kontrolle gut und gut mischen. </li><li> Inkubieren bei Raumtemperatur für 10 Minuten im Dunkeln. </li><li> Messen ABSORBAnce sofort mit einem Platten-Lesegerät mit Filter der Wellenlängen zwischen 520nm und 550nm. </li></ol> Wenn Sie verschiedene Teller / Schale Größen nutzen, verwenden 1/1/1 Lautstärke für jede Lösung und Probenüberstand. Eine violette Farbe wird in der positiven Vertiefungen erscheinen. Ergebnisse mit dem Standard erhalten wird Ihnen helfen, prüfen Sie die Stabilität Ihres Lösungen. 3. Detektion von ROS Aktion: oxidierten Proteinen Eine andere Methode, um die Produktion von ROS zu identifizieren, ist am Ende Ergebnisse durch den Nachweis der Oxidation von Proteinen zu suchen. In der Tat spielt ROS ändern Glutathion, ein Antioxidans, das in den meisten Zellen exprimiert wird und eine schützende Rolle gegen ROS. Nach Oxidation durch ROS, Änderung des reduzierten Glutathion (GSH) Ergebnisse in der Sulfhydryl (Thiol)-Gruppe der Cystein mit einem zweiten Glutathion über eine Disulfidbrücke verbunden. Dies führt zu der Bildung eines dimerisierten Protein (oxidierte Protein GSSG). GSH kann über Modifikation von GSSG durch das Enzym Glutathion-Reduktase wiederhergestellt werden. Der Anstieg des GSSG / GSH-Verhältnis spiegelt oxidativen Stress. Die folgenden Test basiert auf der Detektion und Quantifizierung dieser oxidierten Proteinen. Diese Methode ist nicht für spezifische ROS selektive, sondern erkennt die Auswirkungen von NO, H 2 O 2, O 2 - und andere ROS. Hier messen wir die Gesamtmenge der oxidierten (GSSG) und reduziertem (GSH) Glutathion mit Biolumineszenz-Signale. <ol><li> Seed Ihre Zellen in einer 96-Well-Platte (weiß / transparent, flache Böden) wie gewohnt. Für unser Experiment haben wir 1x10 4 Zellen pro Well für adhärente RAW 264.7 und A549-Zellen und Fahrwerk Jurkat-Zellen. Je nach Größe Ihrer Zellen diese Zahlen weiter angepasst werden kann. Wir empfehlen die Platte anhaftenden Zellen am Tag vor dem Test, damit sie auf die Platte zu befestigen. Genug Vertiefungen sollten für oxidierte und reduzierte Protein-Erkennung sowie für &quot;medium nur&quot; vorbereitet werden undunbehandelte Zellen als Kontrolle. Verwenden Sie dreifach für alle Bedingungen. </li><li> Verwöhnen Sie Ihre Zellen mit Ihrem Medikament für die erforderliche Zeit. Hier haben wir behandelt, Jurkat-Zellen und A549 Zellen für 1h mit H 2 O 2 (bei ​​5 mm und 2,5 mm bzw.). RAW 264.7 Zellen wurden mit 200ng/ml LPS für 16 h behandelt Während dieser Inkubationszeit, bringen alle Ihre Reagenzien auf Raumtemperatur (RT). Nehmen Sie alle Reagenzien nicht mehr als 30 Minuten vor der Durchführung des Tests. Die folgende Tabelle zeigt die Volumina der Reagenzien pro Vertiefung. Wenn möglich, ist es wichtig, die Medien mit dem Reduktionsmittel, bevor Sie mit dem Test zu entfernen. </li></ol><table border="1"><tbody><tr><td rowspan="2"></td><td colspan="2"> Adhärenten Zellen </td><td colspan="2"> Suspensionszellen </td></tr><tr><td> Insgesamt Glu Lysis </td><td> Oxidierte Glu Lysis </td><td> Insgesamt Glu Lysis </td><td> Oxidierte Glu Lysis </strong&gt; </td></tr><tr><td> NEM, 25mm </td><td> keiner </td><td> 0.5μl </td><td> keiner </td><td> 0.5μl </td></tr><tr><td> Luciferin-NT </td><td> 1 ul </td><td> 1 ul </td><td> 1 ul </td><td> 1 ul </td></tr><tr><td> Passive Lysis Buffer, 5X </td><td> 10 &amp;mgr; l </td><td> 10 &amp;mgr; l </td><td> 10 &amp;mgr; l </td><td> 10 &amp;mgr; l </td></tr><tr><td> Destilliertes Wasser </td><td> 39.0μl </td><td> 38.5μl </td><td> 14μl </td><td> 13.5μl </td></tr><tr><td> Das endgültige Volumen pro Well </td><td> 50 ul </td><td> 50 ul </td><td> 25 &amp;mgr; l </td><td> 25 &amp;mgr; l </td></tr></tbody></table><ol start="3"><li> Entfernen Sie Medium aus Brunnen mit adhärenten Zellen. Entfernen Sie nicht mittelfristig in Brunnen mit Suspensionszellen. </li><li> Add reduziertes Glutathion Lyse-Reagenz oder oxidiertem Glutathion Lysereagens in die entsprechenden Wells und schütteln für 5 Minuten bei RT. </li><li> Fügen Sie die Luciferin Generation Reagenz und INCUBATe für 30 Minuten bei RT. </li><li> Fügen Sie die Luciferin Nachweisreagenz und Inkubation für 15 Minuten bei RT. </li><li> Lesen Sie die Biolumineszenz-Signal an Integrationszeit von 0,25-1 Sekunde pro auch mit einem Platten-Lesegerät Luminometer. </li></ol><table border="1"><tbody><tr><td></td><td> Adhärenten Zellen </td><td> Suspensionszellen </td></tr><tr><td> Die Zellzahl pro Well </td><td> 1x10 4 </td><td> 1x10 4 </td></tr><tr><td> Zellsuspension pro Well + Drogen </td><td> 100 ul, vor 3,4 entfernt werden </td><td> 25 &amp;mgr; l, darf nicht entfernt werden </td></tr><tr><td> Reduziertes Glutathion Lysereagens </td><td> 50 pl </td><td> 25 &amp;mgr; l </td></tr><tr><td> Oxidiertem Glutathion Lysereagens </td><td> 50 pl </td><td> 25 &amp;mgr; l </td></tr><tr><td> Luciferin-Generation Reagent </td><td> 50 pl </td><td> 50 pl </td></tr><tr><td> Luciferin Nachweisreagenz </td><td> 100 ul </td><td> 100 ul </td></tr></tbody></table> 4. Repräsentative Ergebnisse: <img alt="Abbildung 1" src="/files/ftp_upload/3357/3357fig1.jpg" /> Abbildung 1: Nachweis von ROS mit Carboxy-H2DCFDA Farbstoff. Jurkat-Zellen (humane Leukämie-Zelllinie) mit H 2 O 2 behandelt wurden, zu nicht behandelten Zellen im Vergleich. ROS induziert die Änderung der Carboxy-H 2 DCFDA, dass grüner fluoresziert wie mittels Durchflusszytometrie, die fluoreszierende Gipfel in H 2 O 2 behandelten Zellen bis zu den Gipfeln in der Kontrollgruppe (H 2 O 2 behandelten Zellen gefärbt mit Oxidations-und Kleinschreibung Farbstoff und nicht im Vergleich Verschiebung erkannt behandelten Zellen mit Carboxy-H 2 DCFDA gebeizt). Die Ergebnisse bestätigen das Vorhandensein von ROS in den behandelten Zellen. <img alt="Abbildung 2" src="/files/ftp_upload/3357/3357fig2.jpg" /> Abbildung 2: Nachweis of NO mit Griess-Reagenzien. RAW 264.7 Zellen (Maus-Makrophagen) wurden mit LPS und IL-4 behandelt. Ein signifikant erhöhtes in NO-Produktion wurde in den behandelten Zellen im Vergleich zu unbehandelten Zellen steuern erkannt. <table border="1"><tbody><tr><td> Zell-Linien </td><td> Unbehandelte </td><td> Behandelte </td></tr><tr><td> RAW 264.7 </td><td> 13,0 </td><td> 8,3 </td></tr><tr><td> A549 </td><td> 21,6 </td><td> 10,5 </td></tr><tr><td> Jurkat </td><td> 5,2 </td><td> 2,8 </td></tr></tbody></table> Tabelle 1: Nachweis von ROS vermittelten Oxidation von Proteinen. RAW 264.7 Zellen mit LPS, Jurkat und A549 behandelt wurden (menschlichen Lungenkrebs-Zellen) wurden mit H 2 O 2 behandelt. Die Ergebnisse werden ausgedrückt als das Verhältnis reduziert (GSH) / oxidiert (GSSG) Glutathion. Niedrigere Verhältnisse von Glutathion (GSH) / (GSSG) wurden in den behandelten Zellen im Vergleich zu erfassendeKontrolle unbehandelte Zellen und enthüllt, dass Proteine ​​mehr wurden in den behandelten Zellen oxidiert.

<ol>
	<li>Guzik, T. J., Korbut, R., Adamek-Guzik, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+and+superoxide+in+inflammation+and+immune+regulation.">Nitric oxide and superoxide in inflammation and immune regulation.</a> J Physiol Pharmacol. 54, 469-487 (2003).</li><li>Perl, A., Gergely, P., Banki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+T+cells+of+patients+with+systemic+lupus+erythematosus.">Mitochondrial dysfunction in T cells of patients with systemic lupus erythematosus.</a> Int Rev Immunol. 23, 293-313 (2004).</li><li>Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T., Mazur, M., Telser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+and+antioxidants+in+normal+physiological+functions+and+human+disease.">Free radicals and antioxidants in normal physiological functions and human disease.</a> Int J Biochem Cell Biol. 39, 44-84 (2007).</li><li>Droge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+in+the+physiological+control+of+cell+function.">Free radicals in the physiological control of cell function.</a> Physiol Rev. 82, 47-95 (2002).</li><li>Nakamura, K., Yube, K., Miyatake, A., Cambier, J. C., Hirashima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+CD4+D3-D4+membrane+proximal+extracellular+domain+for+the+inhibitory+effect+of+oxidative+stress+on+activation-induced+CD4+down-regulation+and+its+possible+role+for+T+cell+activation.">Involvement of CD4 D3-D4 membrane proximal extracellular domain for the inhibitory effect of oxidative stress on activation-induced CD4 down-regulation and its possible role for T cell activation.</a> Mol Immunol. 39, 909-921 (2003).</li><li>Los, M., Droge, W., Stricker, K., Baeuerle, P. A., Schulze-Osthoff, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+as+a+potent+activator+of+T+lymphocyte+functions.">Hydrogen peroxide as a potent activator of T lymphocyte functions.</a> Eur J Immunol. 25, 159-165 (1995).</li><li>Deem, T. L., Cook-Mills, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+cell+adhesion+molecule+1+(VCAM-1)+activation+of+endothelial+cell+matrix+metalloproteinases:+role+of+reactive+oxygen+species.">Vascular cell adhesion molecule 1 (VCAM-1) activation of endothelial cell matrix metalloproteinases: role of reactive oxygen species.</a> Blood. 104, 2385-2393 (2004).</li><li>Pacher, P., Beckman, J. S., Liaudet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+and+peroxynitrite+in+health+and+disease.">Nitric oxide and peroxynitrite in health and disease.</a> Physiol Rev. 87, 315-424 (2007).</li><li>Griendling, K. K., Sorescu, D., Lassegue, B., Ushio-Fukai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+protein+kinase+activity+and+gene+expression+by+reactive+oxygen+species+and+their+role+in+vascular+physiology+and+pathophysiology.">Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology.</a> Arterioscler Thromb Vasc Biol. 20, 2175-2183 (2000).</li><li>Loh, K., Deng, H., Fukushima, A., Cai, X., Boivin, B., Galic, S., Bruce, C., Shields, B. J., Skiba, B., Ooms, L. M., Stepto, N., Wu, B., Mitchell, C. A., Tonks, N. K., Watt, M. J., Febbraio, M. A., Crack, P. J., Andrikopoulos, S., Tiganis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+enhance+insulin+sensitivity.">Reactive oxygen species enhance insulin sensitivity.</a> Cell Metab. 10, 260-272 (2009).</li><li>Goldstein, B. J., Mahadev, K., Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+paradox:+insulin+action+is+facilitated+by+insulin-stimulated+reactive+oxygen+species+with+multiple+potential+signaling+targets.">Redox paradox: insulin action is facilitated by insulin-stimulated reactive oxygen species with multiple potential signaling targets.</a> Diabetes. 54, 311-321 (2005).</li></ol>

Wir erhielten Unterstützung von Promega für diese Veröffentlichung.

Mehrere pathologischen Situationen wie entzündlichen Erkrankungen, Krebs, Ischämie / Reperfusion und auch Behandlungen wie Bestrahlung oder Chemotherapie (dh Cisplatin) induzieren ROS Überproduktion. So erkennen und zu messen ROS Ebenen ist in vielen grundlegenden, präklinischen und klinischen Studien wichtig. Allerdings haben ROS sehr kurze Halbwertszeiten und könnte kompliziert zu erkennen. Hier schlagen wir vor, einfache Tests, die routinemäßig verwendet werden, und weithin akzeptierte für den Nachweis von Pr...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> 5 - (und-6)-Carboxy-2 &#39;, 7&#39;-Dichloro-diacetat (Carboxy-DCFDA) </td><td> Molecular Probes </td><td> C369 </td><td> Steuerung </td></tr><tr><td> Carboxy-H 2 DCFDA </td><td> Molecular Probes </td><td> C400 </td><td></td></tr><tr><td> Sulfanilamid </td><td> Sigma </td><td> S9251-100G </td><td></td></tr><tr><td> N-1-napthylethylenediamine dihydrochlorid </td><td> Sigma </td><td> N9125-10G </td><td></td></tr><tr><td> Nitrit-Standard </td><td> Sigma </td><td> 237213-100G </td><td></td></tr><tr><td> GSH / GSSG-Glo Assay </td><td> Promega </td><td> V6612 </td><td> Um oxidiert, reduziert quantifizieren oder oxidierten / reduzierten Glutathion </td></tr></tbody>
</table>

production and detection of reactive oxygen species (ros) in cancers

Reaktive Sauerstoffspezies sind eine Reihe von Molekülen, die Schäden von DNA und RNA und oxidieren Proteine ​​und Lipide (Lipid peroxydation). Diese reaktiven Moleküle enthalten eine Sauerstoff und sind H 2 O 2 (Wasserstoffperoxid), NO (Stickstoffmonoxid), O 2 - (Oxid-Anionen), Peroxynitrit (ONOO -), hydrochlorous Säure (HOCl) und Hydroxyl-Radikal (OH -) . Oxidativer Arten sind nicht nur unter pathologischen Situationen (Krebs, ischämischer / Reperfusion, neurologischen und Herz-Kreislauf-Erkrankungen, Infektionskrankheiten, Entzündungskrankheiten 1, Autoimmunerkrankungen 2, etc ...), sondern auch während der physiologische (nicht krankhafte) Situationen wie Zellstoffwechsel 3 hergestellt , 4. In der Tat spielen ROS eine wichtige Rolle in vielen zellulären Signalwege (Proliferation, Zell-Aktivierung 5, 6, 7 etc. Migration.). ROS können schädlich sein (es wird dann bezeichnet als&quot;Oxidativen Stress und nitrosative&quot;), wenn in großen Mengen in den intrazellulären Kompartimenten und Zellen produziert allgemein ROS reagieren heraufregulierende Antioxidantien wie Superoxid-Dismutase (SOD) und Katalase (CAT), Glutathionperoxidase (GPx) und Glutathion (GSH), das schützt sie durch die Umwandlung gefährlichen freien Radikale in unschädliche Moleküle (zB Wasser). Vitamine C und E sind auch als ROS-Fänger (Antioxidantien) beschrieben worden. Freie Radikale sind vorteilhaft in niedrigen beträgt 3. Makrophagen und neutrophilen Granulozyten-vermittelte Immunantwort beinhalten die Produktion und Freisetzung von NO, die Viren, Krankheitserreger und Tumorproliferation 8 hemmt. NO reagiert auch mit anderen ROS und somit hat auch eine Rolle als detoxifier (ROS Scavenger). Schließlich NO wirkt auf Schiffen, um den Blutfluss, die wichtig für die Anpassung der Muskel längere Übung 9, 10 zu regulieren. Mehrere Veröffentlichungen haben auch gezeigt, dass ROS in Insulin-sens beteiligt sinditivity 11, 12. Zahlreiche Methoden zur ROS-Produktion zu bewerten sind. In diesem Artikel schlagen wir mehrere einfache, schnelle und kostengünstige Assays; diese Tests wurden von zahlreichen Publikationen validiert und werden routinemäßig verwendet werden, um ROS oder ihre Auswirkungen in Säugetierzellen zu erkennen. Während einige dieser Tests mehrere ROS erkennen, erkennen andere nur ein einziges ROS.

Watch this Scientific Journal Video about Production and Detection of Reactive Oxygen Species ROS in Cancers at JoVE.com

Production and Detection of Reactive Oxygen Species ROS in Cancers

Hier schlagen wir einfache Methoden zu testen und auszuwerten Anwesenheit von reaktiven Sauerstoffspezies in den Zellen.

Produktion und Nachweis von reaktiven Sauerstoffspezies (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

production-and-detection-of-reactive-oxygen-species-ros-in-cancers

Research

JoVE Journal

Medicine

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

69.8K Aufrufe.  Baylor College of Medicine. Hier schlagen wir einfache Methoden zu testen und auszuwerten Anwesenheit von reaktiven Sauerstoffspezies in den Zellen.

Serie: Production and Detection of Reactive Oxygen Species ROS in Cancers

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

Visualisierung und Analyse des Blutflusses und der Sauerstoffverbrauch in hepatischen Mikrozirkulation: Anwendung auf eine akute Hepatitis-Modell

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

Forschung

Medizin

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Cerenkov Lumineszenz Imaging (CLI) for Cancer Therapy Überwachung

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

Isolierung und Verwendung von Rinder Abschnitte A. und V. mesenterica als Bioassay für Vasoaktivität im Dünndarm Testen

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Die Beurteilung Transmissible Spongiforme Enzephalopathie Artgrenzen mit ein<em&gt; In-vitro-</em&gt; Prion Protein-Umwandlungsassay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T &#945;S and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of &#945;SD (disease-associated &#945;S) in these mice, an ELISA was designed to specifically quantify &#945;SD in sick mice. Analysis of the central nervous system in this mouse model showed the presence of &#945;SD mainly in the caudal brain regions and the spinal cord. There were no differences in &#945;SD distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of &#945;SD immunoreactivity using an antibody against Ser129 phosphorylated &#945;S by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of &#945;S. The propagation of &#945;SD, suggesting the involvement of a &#8220;prion-like&#8221; mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

Detection of Disease-associated &amp;#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &amp;#945;-synuclein

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

Nachweis von krankheitsassoziierten α-Synuclein durch verbesserte ELISA im Gehirn von transgenen Mäusen Expression Menschen A53T Mutierte α-Synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

Auslösen Reactive Gliose<em&gt; In Vivo</em&gt; Durch eine Vorderhirn Stab Injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

In Vitro und In Vivo Nachweis von Mitophagy in menschlichen Zellen, C. Elegansund Mäuse

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

Thrombozyten-basierte Erkennung von Stickoxid im Blut durch die Messung VASP Phosphorylierung

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

Quantitative Mapping spezieller Belüftung in der menschlichen Lung mit Proton-Magnetresonanztomographie und Sauerstoff als Kontrastmittel

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When deoxygenated, HbS can polymerize, forming rigid rods of hemoglobin, resulting in the sickling of red blood cells (RBCs). These sickled RBCs have significantly reduced deformability, causing vaso-occlusion, which leads to numerous SCD-related clinical complications, including pain, stroke, and organ damage. RBC deformability is also reduced by RBC dehydration, resulting in dense red blood cells that are more likely to sickle. To date, there is not a single widely available, rapid, and reproducible laboratory assay capable of predicting the disease severity or directly monitoring the treatment effects for novel, non-fetal hemoglobin inducing therapies. In this study, we describe a protocol to measure RBC deformability as a function of pO2 that allows for the quantitation of sickling behavior in SCD patients. Oxygen gradient ektacytometry measures RBC deformability, expressed as the elongation index (EI), as a function of pO2. RBCs are exposed to a fixed shear stress of 30 Pa during one round of deoxygenation and reoxygenation. Six readout parameters are produced. Of these, the point of sickling (PoS), defined as the pO2 at which maximum EI (EImax) shows a 5% decrease, and minimum EI during deoxygenation (EImin) are the most informative, reflecting an individual patient&#8217;s pO2 at which sickling starts and the minimal deformability of a patient&#8217;s red blood cells, respectively. PoS is associated with an individual patient&#8217;s hemoglobin affinity for oxygen, whereas EImin shows a strong correlation with fetal hemoglobin levels. We conclude that oxygen gradient ektacytometry is a promising technique to monitor the treatment of patients with SCD, as a biomarker for anti-sickling agents in clinical and preclinical trials, and an important tool to study sickling behavior of RBCs from individuals with SCD and sickle cell traits.

Here, we present oxygen gradient ektacytometry, a rapid and reproducible method to measure red blood cell deformability in samples from patients with sickle cell disease under controlled deoxygenation and reoxygenation. This technique provides a way to study red blood cell sickling and to monitor sickle cell disease treatment efficacy.

Charakterisierung des Sicklings bei kontrollierter automatisierter Deoxygenierung mit Sauerstoffgradientektacytometrie

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When ...