Name: screening assay for oxidative stress in a feline astrocyte cell line, g355-5
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1. La culture cellulaire d&#39;astrocytes féline et le traitement par H 2 O 2 <ol><li> Lignée cellulaire Culture G355-5 dans un flacon de 75 cm2 avec les médias DMEM plus nutriments à 37 ° C et 5% de CO 2 jusqu&#39;à confluence de 60% ​​(~ 5 jours). Remplacer les médias tous les 1-2 jours ou au besoin pour maintenir une culture saine. </li><li> Retirez le support et ajouter 2 ml de trypsine à 0,25%. Soulevez les cellules par pipetage pour 45 s - 1 min à tempéra...

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Un mécanisme suggère souvent de virus induit des lésions neuronales est le stress oxydatif 1, 4, 7, 9, 13, 15, 16, 18-22, 27, 31. Fondamentalement, il est proposé que l&#39;exposition virale de la glie (astrocytes et microglie) radicaux communiqué réactives de l&#39;oxygène tels que l&#39;hydroxyde, l&#39;anion superoxyde, l&#39;oxyde nitrique, et / ou le peroxyde d&#39;hydrogène, qui sont toxiques pour les neurones. De même, le stress oxydatif a également été suggéré comme un mécanisme majeur ...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> DMEM </td><td> Cellgro </td><td> 15 à 013-CV </td><td></td></tr><tr><td> MEM Vitamines </td><td> Cellgro </td><td> 25 à 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> Acides aminés non essentiels </td><td> Hyclone </td><td> 25 à 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> Acides aminés essentiels </td><td> Cellgro </td><td> 25 à 030 cl </td><td> 0,2% (v / v) </td></tr><tr><td> 500ml système de filtration sous vide </td><td> VWR </td><td> 87006-076 </td><td></td></tr><tr><td> 15 ml tubes coniques </td><td> Falcon </td><td> 352097 </td><td></td></tr><tr><td> 75 culture de tissus cm 2 flacons </td><td> Falcon </td><td> 353136 </td><td></td></tr><tr><td> 6 plaque ainsi la culture de tissus </td><td> Falcon </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> Iodure de propidium (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> Antibiotique HyQ </td><td> Hyclone </td><td> SV30079.01 </td><td> 0,1% (v / v) </td></tr><tr><td> G355-5 cellules </td><td> ATCC </td><td> LCR-2033 </td><td> Normal du cerveau féline </td></tr></tbody></table>

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

Un mécanisme souvent suggéré de virus induit des lésions neuronales est le stress oxydatif. Les astrocytes ont un rôle important dans le contrôle du stress oxydatif du Système Nerveux Central (SNC). Les astrocytes aider à maintenir un environnement homéostatique des neurones ainsi que des neurones à partir de protéger les espèces réactives de l&#39;oxygène (ERO). CM-H2DCFDA est un indicateur de cellules perméables à la présence des ROS. CM-H 2 DCFDA pénètre dans la cellule comme un composé non fluorescent, et devient fluorescent après estérases cellulaires éliminer les groupes acétate, et le composé est oxydé. Le nombre de cellules, mesurée par cytométrie en flux, qui sont jugés fluorescent vert est une indication du nombre de cellules qui sont dans un état d&#39;oxydation. CM-H 2 DCFDA est sensible à l&#39;oxydation par un grand nombre de différents ROS. Ce manque de spécificité, au sujet desquels les ROS peuvent s&#39;oxyder CM-H 2 DCFDA, rend ce composé un régent précieux pour l&#39;utilisation dans les premiers stades d&#39;une enquête pathogenèse, comme ce test peut être utilisé pour dépister un environnement oxydatif cellulaire indépendamment de laquelle l&#39;oxygène radicale ou une combinaison de ROS sont responsables des conditions cellulaires. Une fois qu&#39;il a été établi que les ROS sont présents par oxydation du CM-H 2 DCFDA alors des expériences supplémentaires peuvent être effectuées pour déterminer les ROS ou combinaison de Ross sont impliqués dans le processus de pathogénie particulière. Les résultats de cette étude démontrent que grâce à l&#39;ajout de peroxyde d&#39;hydrogène une augmentation de la CM-H 2 DCFDA fluorescence n&#39;a été détectée par rapport aux contrôles saline, ce qui indique que ce test est un test utile pour détecter un environnement oxydatif dans les cellules G355-5, un féline lignée d&#39;astrocytes.

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

Une méthode de dépistage pour détecter des environnements cellulaires oxydatif est de mesurer l&#39;oxydation de la CM-H2DCFDA. Une fois oxydé dans une cellule, le CM-H2DCFDA changements de la non-fluorescente en un composé fluorescent. Ce changement de fluorescence est mesurée par cytométrie en flux et indique le nombre de cellules dans un milieu oxydant.

Test de dépistage du stress oxydatif dans une lignée d&#39;astrocytes féline, 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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