We thank ReadiSorb Products for their generous support of these studies.

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 containing 10 ml of media. Mix gently.</li>
<li>Centrifuge at 300 x g, 23&deg;C for 3 min to obtain a pellet. Discard the supernatant without disturbing the pellet.</li>
<li>Add 6-7 ml of fresh media and resuspend the cells.</li>
<li>Add &tilde; 1 ml of cells to each well of a 6-well plate. Add 2-3 ml of media to each well and incubate until 80-90% confluent.</li>
<li>Prepare a 100&mu;M solution of peroxide (H2O2) in media.</li>
<li>Remove media from each well and replace with either 2 ml fresh media (control), 2 ml of H2O2 (treatment) or 2ml of DPBS. Incubate at 37&deg;C, 5% CO2 for 3 h.</li>
<li>Remove solutions and add 1 ml of trypsin to lift the cells. Transfer cells to a 1.7 ml Eppendorf tube containing 0.5 ml DPBS. This step should be performed on each well individually in order to prevent cell death from prolonged trypsin incubation.</li>
<li>Spin at 300 x g for 3 min, discard the supernatant and resuspend in 1 ml of DPBS.</li>
</ol>
2. Staining for ROS
<ol>
<li>All steps for staining should be carried out with minimal light exposure to prevent bleaching.</li>
<li>Add 1 &mu;l of 50 mM CCCP to the positive control. Incubate at 37&deg;C, 5% CO2 for 5 min.</li>
<li>Add 5 &mu;l of a 10 mM CM-H2DCFDA solution to the H2O2 group. Incubate at 37&deg;C, 5% CO2 for 15-30 min. No stain is added to the saline sample.</li>
<li>Wash samples with 2 ml of DPBS to remove residual stain.</li>
<li>Spin at 300 x g for 3 min to obtain a pellet.</li>
<li>Resuspend cells in 0.5 ml of DPBS. Add 1 &mu;l PI to each sample, except the saline.</li>
<li>Transfer samples to a 5ml flow cytometer tube.</li>
<li>Run on the flow cytometer.</li>
</ol>
3. Flow cytometry
<ol>
<li>Run samples on a Beckman Coulter FC500 (or equivalent) equipped with a 488 nm argon laser and the following band passes: 525, 775 and 620 nm, all &plusmn; 20 nm.</li>
<li>Prepare the appropriate controls: unstained cells, CM-H2DCFDA stained only, PI only, double stained (negative control).</li>	
<li>Make a protocol that has the following histoplots: FS vs. SS, PI vs. CM-H2DCFDA. It should also have the following histograms: cell number vs. FL1, FL2 and FL3, which corresponds to the stains.</li>
<li>Before running the samples, check equipment, fluids and waste container. Warm up the equipment for at least 20 min.</li>
<li>Clean the equipment and perform a calibration with the appropriate beads according to the manufacturers standards.</li>
<li>Select your protocol and run the samples.</li>
<li>Clean the equipment as done previously.</li>
<li>Clean the vacuum line according to the manufacturers standards.</li>
<li>Turn off the flow and clean the head/vacuum line.</li>
<li>Turn off the software and the computer.</li>
</ol>
4. Representative Results:
Flow cytometry results were analyzed using FlowJo 7.6 and the stain controls were used to objectively set the gating. Based on this, healthy cells appear on the left of the histogram and ROS (oxidative stress) was detected as a shift of cells to the right. Figure 1 shows the results of the effect of hydrogen peroxide on healthy feline astrocytes. Data from FL1 was used to measure the intensity of CM-H2DCFDA, which indicates the presence of ROS. As expected, a higher amount of ROS was detected in the sample treated with H2O2. This is displayed on the histogram as a shift in fluorescent intensity from left to right. When comparing healthy cells treated with DMEM versus DPBS, there was no significant change in the amount of ROS (Fig 2).
<img src="/files/ftp_upload/2841/2841fig1.jpg" alt="Figure 1"/> 
Figure 1. Flow cytometry results of levels of oxidative stress in healthy feline astrocytes and the effect of H2O2 on healthy cells. The results indicate that H2O2 increases levels of ROS in healthy cells as compared to cells with no treatment (DMEM).
<img src="/files/ftp_upload/2841/2841fig2.jpg" alt="Figure 2"/> 
Figure 2. Flow Cytometry results of levels of ROS detected in healthy cells incubated for 3hrs with DPBS.

<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. L., Ali, S., Epstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+oxygen-based+radicals+in+methamphetamine-induced+neurotoxicity:+evidence+from+the+use+of+CuZnSOD+transgenic+mice.">Involvement of oxygen-based radicals in methamphetamine-induced neurotoxicity: evidence from the use of CuZnSOD transgenic mice.</a> Ann N Y Acad Sci. 738, 388-3891 (1994).</li><li>Cathcart, R., Schwiers, E., Ames, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+picomole+levels+of+hydroperoxides+using+a+fluorescent+dichlorofluorescein+assay.">Detection of picomole levels of hydroperoxides using a fluorescent dichlorofluorescein assay.</a> Anal Biochem. 134, 111-116 (1983).</li><li>Chao, C. C., Hu, S., Peterson, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glia:+the+not+so+innocent+bystanders.">Glia: the not so innocent bystanders.</a> J Neurovirol. 2, 234-239 (1996).</li><li>Cooper, A. J., Kristal, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+roles+of+glutathione+in+the+central+nervous+system.">Multiple roles of glutathione in the central nervous system.</a> Biol Chem. 378, 793-802 (1997).</li><li>Cubells, J. F., Rayport, S., Rajendran, G., Sulzer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methamphetamine+neurotoxicity+involves+vacuolation+of+endocytic+organelles+and+dopamine-dependent+intracellular+oxidative+stress.">Methamphetamine neurotoxicity involves vacuolation of endocytic organelles and dopamine-dependent intracellular oxidative stress.</a> J Neurosci. 14, 2260-2271 (1994).</li><li>Dawson, V. L., Dawson, T. M., Uhl, G. R., Snyder, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunodeficiency+virus+type+1+coat+protein+neurotoxicity+mediated+by+nitric+oxide+in+primary+cortical+cultures.">Human immunodeficiency virus type 1 coat protein neurotoxicity mediated by nitric oxide in primary cortical cultures.</a> Proc Natl Acad Sci U S A. 90, 3256-329 (1993).</li><li>Desagher, S., Glowinski, J., Premont, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+protect+neurons+from+hydrogen+peroxide+toxicity.">Astrocytes protect neurons from hydrogen peroxide toxicity.</a> J Neurosci. 16, 2553-2562 (1996).</li><li>Dewhurst, S., Gelbard, H. A., Fine, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathogenesis+of+AIDS.">Neuropathogenesis of AIDS.</a> Mol Med Today. 2, 16-23 (1996).</li><li>Dringen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+and+functions+of+glutathione+in+brain.">Metabolism and functions of glutathione in brain.</a> Prog Neurobiol. 62, 649-671 (2000).</li><li>Dringen, R., Hamprecht, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+restoration+as+indicator+for+cellular+metabolism+of+astroglial+cells.">Glutathione restoration as indicator for cellular metabolism of astroglial cells.</a> Dev Neurosci. 20, 401-407 (1998).</li><li>Dringen, R., Pfeiffer, B., Hamprecht, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+the+Antioxidant+Glutathione+in+Neurons:+Supply+by+Astrocytes+of+CysGly+as+Precursor+for+Neuronal+Glutathione.">Synthesis of the Antioxidant Glutathione in Neurons: Supply by Astrocytes of CysGly as Precursor for Neuronal Glutathione.</a> J Neurosci. 19, 562-569 (1999).</li><li>Everall, I. P. H. u. d. s. o. n., L, R. W. K. e. r. w. i. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+absolute+levels+of+ascorbic+acid+and+unaltered+vasoactive+intestinal+polypeptide+receptor+binding+in+the+frontal+cortex+in+acquired+immunodeficiency+syndrome.">Decreased absolute levels of ascorbic acid and unaltered vasoactive intestinal polypeptide receptor binding in the frontal cortex in acquired immunodeficiency syndrome.</a> Neurosci Lett. 224, 119-1122 (1997).</li><li>Gabriel, C., Camins, A., Sureda, F. X., Aquirre, L., Escubedo, E., Pallas, M., Camarasa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+nitric+oxide+generation+in+mammalian+neurons+using+dichlorofluorescin+diacetate+and+flow+cytometry.">Determination of nitric oxide generation in mammalian neurons using dichlorofluorescin diacetate and flow cytometry.</a> J Pharmacol Toxicol Methods. 38, 93-938 (1997).</li><li>Gendelman, H. E., Genis, P., Jett, M., Zhai, Q. H., Nottet, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+model+system+for+HIV-1-induced+brain+injury.">An experimental model system for HIV-1-induced brain injury.</a> Adv Neuroimmunol. 4, 189-1893 (1994).</li><li>Hayman, M., Arbuthnott, G., Harkiss, G., Brace, H., Filippi, P., Philippon, V., Thomson, D., Vigne, R., Wright, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxicity+of+peptide+analogues+of+the+transactivating+protein+tat+from+Maedi-Visna+virus+and+human+immunodeficiency+virus.">Neurotoxicity of peptide analogues of the transactivating protein tat from Maedi-Visna virus and human immunodeficiency virus.</a> Neuroscience. 53, 1-6 (1993).</li><li>Hirata, H., Ladenheim, B., Rothman, R. B., Epstein, C., Cadet, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methamphetamine-induced+serotonin+neurotoxicity+is+mediated+by+superoxide+radicals.">Methamphetamine-induced serotonin neurotoxicity is mediated by superoxide radicals.</a> Brain Res. , 677-6345 (1995).</li><li>Koka, P., He, K., Zack, J. A., Kitchen, S., Peacock, W., Fried, I., Tran, T., Yashar, S. S., Merrill, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunodeficiency+virus+1+envelope+proteins+induce+interleukin+1,+tumor+necrosis+factor+alpha,+and+nitric+oxide+in+glial+cultures+derived+from+fetal,+neonatal,+and+adult+human+brain.">Human immunodeficiency virus 1 envelope proteins induce interleukin 1, tumor necrosis factor alpha, and nitric oxide in glial cultures derived from fetal, neonatal, and adult human brain.</a> J Exp Med. 182, 941-951 (1995).</li><li>Kong, L. Y., Wilson, B. C., McMillian, M. K., Bing, G., Hudson, P. M., Hong, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+the+HIV-1+envelope+protein+gp120+on+the+production+of+nitric+oxide+and+proinflammatory+cytokines+in+mixed+glial+cell+cultures.">The effects of the HIV-1 envelope protein gp120 on the production of nitric oxide and proinflammatory cytokines in mixed glial cell cultures.</a> Cell Immunol. 172, 77-83 (1996).</li><li>Koutsilieri, E., Gotz, M. E., Sopper, S., Sauer, U., Demuth, M., ter Meulen, V., Riederer, P. <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+glutathione+and+cell+toxicity+following+exposure+to+neurotropic+substances+and+human+immunodeficiency+virus-1+in+vitro.">Regulation of glutathione and cell toxicity following exposure to neurotropic substances and human immunodeficiency virus-1 in vitro.</a> J Neurovirol. 3, 342-349 (1997).</li><li>Lipton, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similarity+of+neuronal+cell+injury+and+death+in+AIDS+dementia+and+focal+cerebral+ischemia:+potential+treatment+with+NMDA+open-channel+blockers+and+nitric+oxide-related+species.">Similarity of neuronal cell injury and death in AIDS dementia and focal cerebral ischemia: potential treatment with NMDA open-channel blockers and nitric oxide-related species.</a> Brain Pathol. 6, 507-517 (1996).</li><li>Lipton, S. A., Yeh, M., Dreyer, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+current+models+of+HIV-related+neuronal+injury:+platelet-activating+factor,+arachidonic+acid+and+nitric+oxide.">Update on current models of HIV-related neuronal injury: platelet-activating factor, arachidonic acid and nitric oxide.</a> Adv Neuroimmunol. 4, 181-188 (1994).</li><li>Mills, E. M., Takeda, K., Yu, Z. X., Ferrans, V., Katagiri, Y., Jiang, H., Lavigne, M. C., Leto, T. L., Guroff, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+growth+factor+treatment+prevents+the+increase+in+superoxide+produced+by+epidermal+growth+factor+in+PC12+cells.">Nerve growth factor treatment prevents the increase in superoxide produced by epidermal growth factor in PC12 cells.</a> J Biol Chem. 273, 22165-22168 (1998).</li><li>Peuchen, S., Duchen, M. R., Clark, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+glutathione+redox+state+in+adult+astrocytes.">Modulation of the glutathione redox state in adult astrocytes.</a> Biochem Soc Trans. 24, 449S-449S (1996).</li><li>Possel, H., Noack, H., Augustin, W., Keilhoff, G., Wolf, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2,7-Dihydrodichlorofluorescein+diacetate+as+a+fluorescent+marker+for+peroxynitrite+formation.">2,7-Dihydrodichlorofluorescein diacetate as a fluorescent marker for peroxynitrite formation.</a> FEBS Lett. 416, 175-178 (1997).</li><li>Sagara, J., Makino, N., Bannai, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+efflux+from+cultured+astrocytes.">Glutathione efflux from cultured astrocytes.</a> J Neurochem. 66, 1876-1881 (1996).</li><li>Stefano, G. B., Smith, E. M., Paemen, L. R., Hughes, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+gp120+associated+neurological+deficits:+a+potential+role+for+nitric+oxide+and+other+signal+molecules.">HIV gp120 associated neurological deficits: a potential role for nitric oxide and other signal molecules.</a> Advances in Neuroimmunology. 3, 47-57 (1993).</li><li>Sundaresan, M., Yu, Z. X., Ferrans, V. J., Irani, K., Finkel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+generation+of+H2O2+for+platelet-derived+growth+factor+signal+transduction.">Requirement for generation of H2O2 for platelet-derived growth factor signal transduction.</a> Science. 270, 296-299 (1995).</li><li>Wang, X. F., Cynader, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+provide+cysteine+to+neurons+by+releasing+glutathione.">Astrocytes provide cysteine to neurons by releasing glutathione.</a> J Neurochem. 74, 1434-1442 (2000).</li><li>Wilson, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidant+defense+of+the+brain:+a+role+for+astrocytes.">Antioxidant defense of the brain: a role for astrocytes.</a> Can J Physiol Pharmacol. 75, 1149-1163 (1997).</li><li>Zenger, E., Collisson, E. W., Barhoumi, R., Burghardt, R. C., Danave, I. R., Tiffany-Castiglioni, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+cytometric+analysis+of+FIV-induced+injury+in+astroglia.">Laser cytometric analysis of FIV-induced injury in astroglia.</a> Glia. 13, 92-100 (1995).</li></ol>

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.

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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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14.4K Views.  Western University of Health Sciences. Метод скрининга для выявления окислительных средах сотовой является измерение окисления CM-H2DCFDA. Как только окисленный в клетке, CM-H2DCFDA изменениями от не-флуоресцентный в флуоресцентным соединением. Это изменение в флуоресценции измеряют с помощью проточной ?...

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

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major immune cells and only phagocytes in the brain, recent studies have shown that astrocytes also participate in various phagocytic processes, such as developmental synapse elimination and clearance of amyloid beta plaques in Alzheimer&#39;s disease (AD). Despite these findings, the efficiency of astrocyte engulfment and degradation of their targets is unclear compared with that of microglia. This lack of information is mostly due to the lack of an assay system in which the kinetics of astrocyte- and microglia-mediated phagocytosis are easily comparable. To achieve this goal, we have developed a long-term live-imaging in vitro phagocytosis assay to evaluate the phagocytic capacity of purified astrocytes and microglia. In this assay, real-time detection of engulfment and degradation is possible using pH indicator-conjugated synaptosomes, which emit bright red fluorescence in acidic organelles, such as lysosomes. Our novel assay provides simple and effective detection of phagocytosis through live-imaging. In addition, this in vitro phagocytosis assay can be used as a screening platform to identify chemicals and compounds that can enhance or inhibit the phagocytic capacity of astrocytes. As synaptic pruning malfunction and pathogenic protein accumulation have been shown to cause mental disorders or neurodegenerative diseases, chemicals and compounds that modulate the phagocytic capacity of glial cells should be helpful in treating various neurological disorders.

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

This protocol presents an in vitro live-imaging phagocytosis assay to measure the phagocytic capacity of astrocytes. Purified rat astrocytes and microglia are used along with pH indicator-conjugated synaptosomes. This method can detect real-time engulfment and degradation kinetics and provides a suitable screening platform to identify factors modulating astrocyte phagocytosis.

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other proteins associated with neurodegenerative diseases, the reported in vitro aggregation kinetics for tau protein are less consistent presenting a relatively high variability. Here we describe the development of an in vitro aggregation assay that mimics the expected steps associated with tau misfolding and aggregation in vivo. The assay uses the longest tau isoform (huTau441) which contains both N-terminal acidic inserts as well as four microtubule binding domains (MBD). The in vitro aggregation is triggered by addition of heparin and followed continuously by thioflavin T fluorescence in a 96 well microplate format. The tau aggregation assay is highly reproducible between different wells, experimental runs and batches of the protein. The aggregation leads to tau PHF-like morphology which is very efficient in seeding the formation of de novo fibrillar structures. In addition to its application in studying the mechanism of tau misfolding and aggregation, the current assay is a robust tool for screening drugs that could interfere with the pathogenesis of tau.

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

The tau aggregation assay described in this manuscript mimics the anticipated features of in vivo tau misfolding and aggregation.

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...

Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections

Astrocytes possess an astounding degree of morphological complexity that enables them to interact with nearly every type of cell and structure within the brain. Through these interactions, astrocytes actively regulate many critical brain functions, including synapse formation, neurotransmission, and ion homeostasis. In the rodent brain, astrocytes grow in size and complexity during the first three postnatal weeks and establish distinct, non-overlapping territories to tile the brain. This protocol provides an established method for analyzing astrocyte territory volume and astrocyte tiling using free-floating tissue sections from the mouse brain. First, this protocol describes the steps for tissue collection, cryosectioning, and immunostaining of free-floating tissue sections. Second, this protocol describes image acquisition and analysis of astrocyte territory volume and territory overlap volume, using commercially available image analysis software. Lastly, this manuscript discusses the advantages, important considerations, common pitfalls, and limitations of these methods. This protocol requires brain tissue with sparse or mosaic fluorescent labeling of astrocytes, and is designed to be used with common lab equipment, confocal microscopy, and commercially available image analysis software.

This protocol describes methods for sectioning, staining, and imaging free-floating tissue sections of the mouse brain, followed by a detailed description of the analysis of astrocyte territory volume and astrocyte territory overlap or tiling.

Astrocytes possess an astounding degree of morphological complexity that enables them to interact with nearly every type of cell and structure within the ...