Name: screening assay for oxidative stress in a feline astrocyte cell line, g355-5
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1. Cellkulturer av katt astrocyter och behandling med H 2 O 2 <ol><li> Kultur cellinje G355-5 i en 75 cm 2-kolv med DMEM Media Plus näringsämnen vid 37 ° C och 5% CO 2 till 60% konfluenta (~ 5 dagar). Byt media varje 1-2 dagar eller som behövs för att upprätthålla en sund kultur. </li><li> Ta bort media och tillsätt 2 ml 0,25% trypsin. Lyft cellerna genom att pipettera till 45 s - 1 min vid rumstemperatur (RT). </li><li> Snabbt överföra suspensionen till ett 15 ml ...

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En ofta föreslagna mekanismen för att viruset orsakat neuronal skada är oxidativ stress 1, 4, 7, 9, 13, 15, 16, 18-22, 27, 31. I grunden är det föreslås att genom viral exponering Glia (astrocyter och mikroglia) släpp reaktiva syreradikaler som, hydroxid, superoxid anjon, kväveoxid och / eller väteperoxid, som är giftiga för nervceller. Likaså har oxidativ stress också föreslagits som en viktig patologiska mekanism i metamfetamin-inducerad neurotoxicitet 2, 6, 17. Eftersom vår grupp ...

<table border="1"><tbody><tr><th> Reagensets namn </th><th> Företag </th><th> Katalognummer </th><th> Kommentarer (frivilligt) </th></tr><tr><td> DMEM </td><td> Cellgro </td><td> 15-013-CV </td><td></td></tr><tr><td> MEM Vitaminer </td><td> Cellgro </td><td> 25-020-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> Icke essentiella aminosyror </td><td> Hyclone </td><td> 25-025-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> Essentiella aminosyror </td><td> Cellgro </td><td> 25-030-cl </td><td> 0,2% (v / v) </td></tr><tr><td> 500ml vakuumfiltrering systemet </td><td> VWR </td><td> 87006-076 </td><td></td></tr><tr><td> 15 ml koniska rör </td><td> Falcon </td><td> 352.097 </td><td></td></tr><tr><td> 75 cm 2 vävnadskultur kolvar </td><td> Falcon </td><td> 353.136 </td><td></td></tr><tr><td> 6 väl vävnadskultur tallrik </td><td> Falcon </td><td> 353.224 </td><td></td></tr><tr><td> CM-H 2 DCFDA </td><td> Invitrogen </td><td> C6827 </td><td></td></tr><tr><td> Propidiumjodid (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 celler </td><td> ATCC </td><td> CRL-2033 </td><td> Normal kattdjur hjärnan </td></tr></tbody></table>

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

En ofta föreslagna mekanismen för viruset orsakat neuronal skada är oxidativ stress. Astrocyter har en viktig roll i kontrollen oxidativ stress i centrala nervsystemet (CNS). Astrocyter bidra till att upprätthålla en homeostatic miljö för nervceller samt att skydda nervceller från reaktiva syreradikaler (ROS). CM-H2DCFDA är en cell-genomsläpplig indikator för förekomsten av ROS. CM-H 2 DCFDA in i cellen som ett icke-fluorescerande förening, och blir fluorescerande efter cellulära esteraser bort acetat grupper och substansen oxideras. Antalet celler, mätt med flödescytometri, som befinns vara gröna fluorescerande är en indikation på antalet celler som är i en oxidativ tillstånd. CM-H 2 DCFDA är känsligt för oxidation av ett stort antal olika ROS. Denna brist på specificitet om vilka ROS kan oxidera CM-H 2 DCFDA, gör denna förening en värdefull regent för användning i de tidiga stadierna av en patogenes undersökning, eftersom detta test kan användas för att screena för en oxidativ cellulär miljö oavsett vilken syre radikala eller kombinationer av ROS är ansvariga för den cellulära förhållanden. När det har konstaterats att ROS är närvarande genom oxidation av CM-H 2 DCFDA, sedan ytterligare experiment kan utföras för att avgöra vilka ROS eller en kombination av Ross är involverade i synnerhet patogenes processen. Resultaten från denna studie visar att med tillägg av väteperoxid en ökning av CM-H 2 DCFDA fluorescerar upptäcktes i förhållande till saltlösning kontroller, vilket tyder på att denna analys är ett värdefullt test för att upptäcka en oxidativ miljö inom G355-5 celler, en kattdjur astrocyternas cellinje.

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

En screeningmetod för att upptäcka oxidativ cellulära miljöer är att mäta oxidation av CM-H2DCFDA. När oxideras i en cell, CM-H2DCFDA förändringar från icke-fluorescerande i en fluorescerande förening. Denna förändring i fluorescens mäts med flödescytometri och anger antalet celler i en oxidativ miljö.

Screening-analys för oxidativ stress i ett felint astrocyternas 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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