Name: screening assay for oxidative stress in a feline astrocyte cell line, g355-5
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1. Zellkultur von feline Astrozyten und die Behandlung mit H 2 O 2 <ol><li> Kultur-Zelllinie G355-5 in eine 75 cm 2-Kolben mit DMEM-Medium plus Nährstoffe bei 37 ° C und 5% CO 2 bis zu 60% konfluent (~ 5 Tage). Ersetzen Sie die Medien alle 1-2 Tage oder bei Bedarf auf eine gesunde Kultur aufrecht zu erhalten. </li><li> Entfernen Sie das Medium und 2 ml 0,25% Trypsin. Heben Sie die Zellen durch Pipettieren für 45 s - 1 min bei Raumtemperatur (RT). </li><li> Schnelle Übert...

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Ein häufig vorgeschlagene Mechanismus der Virus-induzierten neuronalen Schädigung ist oxidativer Stress 1, 4, 7, 9, 13, 15, 16, 18-22, 27, 31. Grundsätzlich wird vorgeschlagen, dass durch virale Belastung der Glia (Astrozyten und Mikroglia) Freisetzung reaktiver Sauerstoff-Radikale, wie Hydroxid, Superoxidanion, Stickstoffmonoxid und / oder Wasserstoffperoxid, die giftig für Nervenzellen sind. Ebenso hat oxidativer Stress auch als ein wichtiger Pathomechanismus in Methamphetamin-induzierte Neurotoxizität...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> DMEM </td><td> CellGro </td><td> 15 bis 013-CV </td><td></td></tr><tr><td> MEM Vitamine </td><td> CellGro </td><td> 25 bis 020-cl </td><td> 1% (v / v) </td></tr><tr><td> L-Glutamin </td><td> Hyclone </td><td> 17-605E </td><td> 1% (v / v) </td></tr><tr><td> Nicht-essentiellen Aminosäuren </td><td> Hyclone </td><td> 25 bis 025-cl </td><td> 1% (v / v) </td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30071.03 </td><td> 20% (v / v) </td></tr><tr><td> Essentielle Aminosäuren </td><td> CellGro </td><td> 25 bis 030-cl </td><td> 0,2% (v / v) </td></tr><tr><td> 500ml Vakuumfiltration System </td><td> VWR </td><td> 87006-076 </td><td></td></tr><tr><td> 15 ml konische Röhrchen </td><td> Falke </td><td> 352097 </td><td></td></tr><tr><td> 75 cm 2 Zellkulturflaschen </td><td> Falke </td><td> 353136 </td><td></td></tr><tr><td> 6 gut Gewebekulturplatte </td><td> Falke </td><td> 353224 </td><td></td></tr><tr><td> CM-H 2 DCFDA </td><td> Invitrogen </td><td> C6827 </td><td></td></tr><tr><td> Propidiumiodid (PI) </td><td> Sigma </td><td> P4170-10mg </td><td></td></tr><tr><td> Trypsin </td><td> Lonza </td><td> 17-160F </td><td></td></tr><tr><td> H 2 O 2 </td><td> Sigma </td><td> H6520 </td><td></td></tr><tr><td> HyQ Antibiotika </td><td> Hyclone </td><td> SV30079.01 </td><td> 0,1% (v / v) </td></tr><tr><td> G355-5-Zellen </td><td> ATCC </td><td> CRL-2033 </td><td> Normale Katzen Gehirn </td></tr></tbody></table>

screening assay for oxidative stress in a feline astrocyte cell line, g355-5

Eine häufig vorgeschlagene Mechanismus der Virus-induzierten neuronalen Schädigung ist oxidativer Stress. Astrozyten spielen eine wichtige Rolle bei der Kontrolle von oxidativem Stress des Zentralen Nervensystems (ZNS). Astrozyten zur Aufrechterhaltung eines homöostatischen Umgebung für Neuronen sowie Schutz der Neuronen von Reactive Oxygen Species (ROS). CM-H2DCFDA ist ein zellpermeablen Indikator für die Anwesenheit von ROS. CM-H 2 DCFDA in die Zelle als nicht-fluoreszierende Verbindung, und wird fluoreszierende nach zelluläre Esterasen entfernen Sie die Acetat-Gruppen, und die Verbindung wird oxidiert. Die Anzahl der Zellen, mittels Durchflusszytometrie gemessen, dass gefunden zu grün fluoreszierenden sind ein Indiz für die Anzahl der Zellen, die in einer oxidativen Zustand sind. CM-H 2 DCFDA ist anfällig für eine Oxidation durch eine große Anzahl verschiedener ROS. Dieser Mangel an Spezifität in Bezug auf die ROS können CM-H 2 DCFDA oxidieren, macht diese Verbindung eine wertvolle Regenten für den Einsatz in den frühen Phasen eines Pathogenese Untersuchung, da dieser Test kann auf dem Bildschirm für eine oxidative zellulären Umgebung, unabhängig von der Sauerstoff verwendet werden Radikal oder Kombination von ROS sind verantwortlich für die zelluläre Bedingungen. Nachdem festgestellt wurde, dass ROS Gegenwart durch Oxidation von CM-H 2 DCFDA, dann weitere Experimente durchgeführt werden, um festzustellen, welche ROS oder eine Kombination von Ross in der jeweiligen Pathogenese beteiligt sind, sind. Die Ergebnisse dieser Studie zeigen, dass mit der Zugabe von Wasserstoffperoxid eine Erhöhung der CM-H 2 DCFDA fluoreszieren wurde gegenüber dem Kochsalzkontrollen, entdeckt darauf hinweist, dass dieser Test ein wertvolles Test zum Nachweis einer oxidativen Umgebung innerhalb G355-5-Zellen, einer ist feline Astrozyten-Zelllinie.

Watch this Scientific Journal Video about Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5 at JoVE.com

Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5

Ein Screening-Verfahren zur oxidativen zellulären Umgebungen erkennen ist, um die Oxidation von CM-H2DCFDA messen. Sobald in einer Zelle, CM-H2DCFDA Änderungen von nicht-fluoreszierenden in einen fluoreszierenden Verbindung oxidiert. Diese Änderung in der Fluoreszenz wird mittels Durchflusszytometrie gemessen und gibt die Anzahl der Zellen in einer oxidativen Umgebung.

Screening Assay für oxidativen Stress in einem Feline Astrocyte Cell Line, G355-5

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of ...

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