Name: screening assay for oxidative stress in a feline astrocyte cell line, g355-5
Uploaded: 2011-07-13T13:00:00
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1. Cell culture of feline astrocytes and treatment with H2O2
<ol>
<li>Culture cell line G355-5 in a 75 cm2 flask with DMEM media plus nutrients at 37&deg;C and 5% CO2 until 60% confluent (&tilde; 5 days). Replace the media every 1-2 days or as needed to maintain a healthy culture.</li>
<li>Remove media and add 2 ml of 0.25% trypsin. Lift the cells by pipetting for 45 s - 1 min at room temperature (RT).</li> 
<li>Quickly transfer the suspension to a 15 ml conical tube contain...

<ol>
	<li>Bukrinsky, M. I., Nottet, H. S., Schmidtmayerova, H. L., Dubrovsky, H., Flanagan, C. R., Mullins, M. E., Lipton, S. A., Gendelman, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+nitric+oxide+synthase+activity+in+human+immunodeficiency+virus+type+1+(HIV-1)-infected+monocytes:+implications+for+HIV-associated+neurological+disease.">Regulation of nitric oxide synthase activity in human immunodeficiency virus type 1 (HIV-1)-infected monocytes: implications for HIV-associated neurological disease.</a> J Exp Med. 181, 735-745 (1995).</li><li>Cadet, J. 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An often suggested mechanism of virus induced neuronal damage is oxidative stress 1, 4, 7, 9, 13, 15, 16, 18-22, 27, 31. Basically, it is proposed that through viral exposure the glia (astrocytes and microglia) release reactive oxygen radicals such as, hydroxide, superoxide anion, nitric oxide, and/or hydrogen peroxide, which are toxic to neurons. Similarly, oxidative stress has also been suggested as a major pathologic mechanism in methamphetamine-induced neurotoxicity 2, 6, 17. Since our group i...

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 	<td>DMEM</td>
 <td>Cellgro</td>
 <td>15-013-CV</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>MEM Vitamins</td>
 <td>Cellgro</td>
 <td>25-020-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>Non Essential Amino Acids</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>Essential Amino Acids</td>
 <td>Cellgro</td>
 <td>25-030-cl</td>
 <td>0.2% (v/v)</td>
 </tr>
 <tr>
 	<td>500ml vacuum filtration system</td>
 <td>VWR</td>
 <td>87006-076</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>15 ml conical tubes</td>
 <td>Falcon</td>
 <td>352097</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>75 cm2 tissue culture flasks</td>
 <td>Falcon</td>
 <td>353136</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>6 well tissue culture plate</td>
 <td>Falcon</td>
 <td>353224</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>CM-H2DCFDA</td>
 <td>Invitrogen</td>
 <td>C6827</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Propidium Iodide (PI)</td>
 <td>Sigma</td>
 <td>P4170-10mg</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Trypsin</td>
 <td>Lonza</td>
 <td>17-160F</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>H2O2</td>
 <td>Sigma</td>
 <td>H6520</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>HyQ Antibiotic</td>
 <td>Hyclone</td>
 <td>SV30079.01</td>
 <td>0.1% (v/v)</td>
 </tr>
 <tr>
 	<td>G355-5 cells</td>
 <td>ATCC</td>
 <td>CRL-2033</td>
 <td>Normal feline brain</td>
 </tr> 
 </tbody>
</table>

screening assay for oxidative stress in a 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 the Central Nervous System (CNS). Astrocytes help maintain a homeostatic environment for neurons as well as protecting neurons from Reactive Oxygen Species (ROS). CM-H2DCFDA is a cell-permeable indicator for the presence of ROS. CM-H2DCFDA enters the cell as a non-fluorescent compound, and becomes fluorescent after cellular esterases remove the acetate groups, and the compound is oxidized. The number of cells, measured by flow cytometry, that are found to be green fluorescing is an indication of the number of cells that are in an oxidative state. CM-H2DCFDA is susceptible to oxidation by a large number of different ROS. This lack of specificity, regarding which ROS can oxidize CM-H2DCFDA, makes this compound a valuable regent for use in the early stages of a pathogenesis investigation, as this assay can be used to screen for an oxidative cellular environment regardless of which oxygen radical or combination of ROS are responsible for the cellular conditions. Once it has been established that ROS are present by oxidation of CM-H2DCFDA, then additional experiments can be performed to determine which ROS or combination of ROSs are involved in the particular pathogenesis process. The results of this study demonstrate that with the addition of hydrogen peroxide an increase in CM-H2DCFDA fluoresce was detected relative to the saline controls, indicating that this assay is a valuable test for detecting an oxidative environment within G355-5 cells, a feline astrocyte cell line.

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

A screening method to detect oxidative cellular environments is to measure the oxidation of CM-H2DCFDA. Once oxidized within a cell, CM-H2DCFDA changes from non-fluorescent into a fluorescent compound. This change in fluorescence is measured by flow cytometry and indicates the number of cells in an oxidative environment.

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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