Name: screening assay for oxidative stress in a feline astrocyte cell line, g355-5
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1. Cel cultuur van katachtige astrocyten en de behandeling met H 2 O 2 <ol><li> Cultuur cellijn G355-5 in een 75 cm 2 kolf met DMEM media plus voedingsstoffen bij 37 ° C en 5% CO 2 tot 60% confluent (~ 5 dagen). Vervang de media om de 1-2 dagen of als nodig is om een ​​gezonde cultuur te behouden. </li><li> Verwijder de media en voeg 2 ml van 0,25% trypsine. Til de cellen door pipetteren voor 45 s - 1 min bij kamertemperatuur (RT). </li><li> Snel overbrengen van de scho...

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Een vaak voorgestelde mechanisme van het virus geïnduceerde neuronale schade is oxidatieve stress 1, 4, 7, 9, 13, 15, 16, 18-22, 27, 31. In principe, wordt voorgesteld dat door middel van virale blootstelling aan de glia (astrocyten en microglia) release reactieve zuurstofradicalen, zoals, hydroxide, superoxide anion, stikstofoxide, en / of waterstof peroxide, die giftig zijn voor neuronen. Ook heeft oxidatieve stress ook voorgesteld als een belangrijke pathologische mechanisme in methamphetamine-geïnduceer...

<table border="1"><tbody><tr><th> Naam van het serum </th><th> Vennootschap </th><th> Catalogus nummer </th><th> Commentaar (optioneel) </th></tr><tr><td> DMEM </td><td> Cellgro </td><td> 15-013-CV </td><td></td></tr><tr><td> MEM Vitaminen </td><td> Cellgro </td><td> 25 tot 020-cl </td><td> 1% (v / v) </td></tr><tr><td> L-glutamine </td><td> Hyclone </td><td> 17-605E </td><td> 1% (v / v) </td></tr><tr><td> Niet-essentiële aminozuren </td><td> Hyclone </td><td> 25 tot 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> Essentiële Aminozuren </td><td> Cellgro </td><td> 25 tot 030-cl </td><td> 0,2% (v / v) </td></tr><tr><td> 500ml vacuümfiltratie systeem </td><td> VWR </td><td> 87006-076 </td><td></td></tr><tr><td> 15 ml conische buizen </td><td> Valk </td><td> 352097 </td><td></td></tr><tr><td> 75 cm 2 weefselkweekflessen </td><td> Valk </td><td> 353136 </td><td></td></tr><tr><td> 6 goed weefselkweek plaat </td><td> Valk </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> Propidiumjodide (PI) </td><td> Sigma </td><td> P4170-10mg </td><td></td></tr><tr><td> Trypsine </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 antibiotica </td><td> Hyclone </td><td> SV30079.01 </td><td> 0,1% (v / v) </td></tr><tr><td> G355-5-cellen </td><td> ATCC </td><td> CRL-2033 </td><td> Normale kat hersenen </td></tr></tbody></table>

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

Een vaak geopperd mechanisme van virus geïnduceerde neuronale schade is oxidatieve stress. Astrocyten hebben een belangrijke rol in het controleren van oxidatieve stress van het centrale zenuwstelsel (CZS). Astrocyten helpen handhaven van een homeostatische omgeving voor neuronen als het beschermen van neuronen van reactive oxygen species (ROS). CM-H2DCFDA is een cel-permeabele indicator voor de aanwezigheid van ROS. CM-H 2 DCFDA komt in de cel als een niet-fluorescerende verbinding, en wordt fluorescerende na cellulaire esterases verwijder de acetaat groepen, en de verbinding is geoxideerd. Het aantal cellen, gemeten door middel van flowcytometrie, die worden gevonden om groene fluorescerende worden is een indicatie van het aantal cellen die in een oxidatieve toestand. CM-H 2 DCFDA is gevoelig voor oxidatie door een groot aantal verschillende ROS. Dit gebrek aan specificiteit, over welke ROS kunnen CM-H 2 DCFDA oxideren, maakt deze verbinding een waardevolle regent voor gebruik in de vroege stadia van een pathogenese onderzoek, omdat deze test kan worden gebruikt om te screenen op een oxidatieve cellulaire omgeving, ongeacht welke zuurstof radicaal of combinatie van ROS zijn verantwoordelijk voor de cellulaire condities. Zodra is vastgesteld dat ROS aanwezig zijn door oxidatie van CM-H 2 DCFDA, dan is extra experimenten kunnen worden uitgevoerd om te bepalen welke ROS of combinatie van Ross zijn betrokken bij de pathogenese bijzonder proces. De resultaten van deze studie tonen aan dat met de toevoeging van waterstofperoxide een toename van de CM-H 2 DCFDA fluoresceren was ten opzichte van de zoutoplossing controles aangetroffen, wat aangeeft dat deze test is een waardevolle test voor het detecteren van een oxidatieve omgeving binnen G355-5-cellen, een katachtige astrocyten cellijn.

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

Een screening methode om oxidatieve cellulaire omgevingen te detecteren is het meten van de oxidatie van CM-H2DCFDA. Zodra geoxideerd in een cel, CM-H2DCFDA veranderingen ten opzichte van niet-fluorescerende in een fluorescerende verbinding. Deze verandering in fluorescentie wordt gemeten door middel van flowcytometrie en geeft het aantal cellen in een oxidatieve omgeving.

Screening Assay voor oxidatieve stress in een Feline astrocyten cellijn, 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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