Agradecemos Produtos ReadiSorb por seu generoso apoio desses estudos.

1. Cultura de células de astrócitos felino e tratamento com H 2 O 2 <ol><li> Linha de cultura de células G355-5 em um balão de 75 cm 2 com a mídia DMEM acrescido de nutrientes, a 37 ° C e 5% de CO 2 até confluentes 60% (~ 5 dias). Substituir a mídia a cada 1-2 dias ou quando necessário para manter uma cultura saudável. </li><li> Remova a mídia e adicionar 2 ml de tripsina 0,25%. Levante as células por pipetagem por 45 s - 1 min à temperatura ambiente (RT). </li><li> Rapidamente transferir a suspensão para um tubo cônico de 15 ml contendo 10 ml de mídia. Misture delicadamente. </li><li> Centrifugar a 300 xg, 23 ° C por 3 min para obter uma pelota. Desprezar o sobrenadante sem perturbar o sedimento. </li><li> Adicionar ml 6-7 de mídia fresco e ressuspender as células. </li><li> Adicionar ~ 1 ml de células para cada poço de uma placa de seis poços. Adicionar 2-3 ml de mídia para cada poço e incubar até confluentes 80-90%. </li><li> Prepare uma solução 100μM de peróxido (H 2 O 2) na mídia. </li><li> Remova a mídia de cada bem e substituir com 2 ml de mídia fresco (controle), 2 ml de H 2 O 2 (tratamento) ou 2 ml de DPBS. Incubar a 37 ° CO C, 5 2% para 3 h. </li><li> Remover soluções e adicionar 1 ml de tripsina para levantar as células. Transferência de células para um tubo Eppendorf 1,7 ml contendo 0,5 ml DPBS. Esta etapa deve ser realizada em cada poço individualmente, a fim de evitar a morte celular de incubação de tripsina prolongada. </li><li> Giram a 300 xg por 3 min, descartar o. Sobrenadante e ressuspender em 1 ml de DPBS </li></ol> 2. Coloração de ROS <ol><li> Todos os passos para a coloração deve ser realizada com a exposição à luz mínima para prevenir branqueamento. </li><li> Adicionar 1 ml de 50 mM CCCP ao controle positivo. Incubar a 37 ° C, 5% CO 2 por 5 min. </li><li> Adicionar 5 mL de uma solução de 10 mM CM-H DCFDA 2 à H 2 O 2 grupo. Incubar a 37 ° C, 5% CO 2 por 15-30 min. Sem mancha é adicionado à amostra salina. </li><li> Lavar as amostras com 2 ml de DPBS para remover mancha residual. </li><li> Giram a 300 xg por 3 min para obter uma pelota. </li><li> Ressuspender as células em 0,5 ml de DPBS. Adicionar 1 mL PI para cada amostra, com exceção do soro fisiológico. </li><li> Transferência de amostras para um tubo de 5ml de fluxo citómetro. </li><li> Executar no citômetro de fluxo. </li></ol> 3. Citometria de fluxo <ol><li> Executar amostras em um Beckman Coulter FC500 (ou equivalente) equipado com um laser de argônio 488 nm ea banda passa a seguir: 525, 775 e 620 nm, todas ± 20 nm. </li><li> Prepare os controles apropriados: não coloridas, CM-H 2 DCFDA manchada apenas, PI só, manchado dupla (controle negativo). </li><li> Faça um protocolo que tem o histoplots seguinte: FS vs SS, PI vs CM-H 2 DCFDA. Ele também deve ter os histogramas seguintes: número de células versus FL1, FL2 e FL3, o que corresponde às manchas. </li><li> Antes de executar as amostras, verificar o equipamento, fluidos e recipiente de resíduos. Aquecer o equipamento para, pelo menos, 20 min. </li><li> Limpar o equipamento e realizar uma calibração com as contas apropriadas de acordo com os padrões dos fabricantes. </li><li> Selecione seu protocolo e executar os exemplos. </li><li> Limpar o equipamento, como feito anteriormente. </li><li> Limpar a linha de vácuo acordo com as normas dos fabricantes. </li><li> Desligue o fluxo e limpar a linha da cabeça / vácuo. </li><li> Desligue o software eo computador. </li></ol> 4. Resultados representativos: Resultados de citometria de fluxo foram analisados ​​utilizando programa FlowJo 7,6 e os controles mancha foram usados ​​para objetivamente definir o gating. Com base nisso, as células saudáveis ​​aparecem à esquerda do histograma e ROS (estresse oxidativo) foi detectado como uma mudança de células para a direita. A Figura 1 mostra os resultados do efeito do peróxido de hidrogênio em astrócitos saudáveis ​​felino. Dados de FL1 foi usado para medir a intensidade da DCFDA 2 CM-H, que indica a presença de ROS. Como esperado, uma maior quantidade de ROS foi detectado na amostra tratada com H 2 O 2. Isso é exibido no histograma como uma mudança na intensidade de fluorescência da esquerda para a direita. Ao comparar as células saudáveis ​​tratados com DMEM versus DPBS, não houve mudança significativa na quantidade de ROS (Fig. 2). <img src="/files/ftp_upload/2841/2841fig1.jpg" alt="Figura 1"/> Figura 1. Fluxo de resultados de citometria de níveis de estresse oxidativo em astrócitos saudáveis ​​felina eo efeito do H 2 O 2 em células saudáveis. Os resultados indicam que H 2 O 2 aumenta os níveis de ROS em células saudáveis, em comparação com as células sem tratamento (DMEM). <img src="/files/ftp_upload/2841/2841fig2.jpg" alt="Figura 2"/> Figura 2. Fluxo Citometria de resultados de níveis de ROS detectado em células saudáveis ​​incubados por 3 horas com 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>

Um mecanismo muitas vezes sugerido de vírus danos induzidos neuronal é o estresse oxidativo 1, 4, 7, 9, 13, 15, 16, 18-22, 27, 31. Basicamente, propõe-se que através da exposição viral da glia (astrócitos e microglia) liberação de radicais de oxigênio reativo, tais como, hidróxido, ânion superóxido, óxido nítrico, e / ou peróxido de hidrogênio, que são tóxicos para os neurônios. Da mesma forma, o estresse oxidativo também tem sido sugerido como um mecanismo patológico em grandes metanfet...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> DMEM </td><td> Cellgro </td><td> 15-013-CV </td><td></td></tr><tr><td> MEM Vitaminas </td><td> Cellgro </td><td> 25-020-cl </td><td> 1% (v / v) </td></tr><tr><td> L-glutamina </td><td> Hyclone </td><td> 17-605E </td><td> 1% (v / v) </td></tr><tr><td> Não Aminoácidos Essenciais </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> Aminoácidos Essenciais </td><td> Cellgro </td><td> 25-030-cl </td><td> 0,2% (v / v) </td></tr><tr><td> Sistema de filtragem a vácuo 500ml </td><td> VWR </td><td> 87006-076 </td><td></td></tr><tr><td> 15 ml tubos cônicos </td><td> Falcão </td><td> 352097 </td><td></td></tr><tr><td> 75 centímetros de cultura de tecidos 2 frascos </td><td> Falcão </td><td> 353136 </td><td></td></tr><tr><td> 6 placa de cultura de tecido bem </td><td> Falcão </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> Iodeto de propídio (PI) </td><td> Sigma </td><td> P4170-10mg </td><td></td></tr><tr><td> Tripsina </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> Antibiótico HyQ </td><td> Hyclone </td><td> SV30079.01 </td><td> 0,1% (v / v) </td></tr><tr><td> G355-5 células </td><td> ATCC </td><td> CRL-2033 </td><td> Cérebro felino normal </td></tr></tbody></table>

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

Um mecanismo muitas vezes sugerido de vírus danos induzidos neuronal é o estresse oxidativo. Astrócitos têm um papel importante no controle do estresse oxidativo do Sistema Nervoso Central (SNC). Astrócitos ajudar a manter um ambiente homeostático para os neurônios, assim como proteger os neurônios de Espécies Reativas de Oxigênio (ROS). CM-H2DCFDA é um indicador de células-permeável para a presença de ROS. CM-H 2 DCFDA entra na célula como um composto não-fluorescente, e se torna fluorescente após esterases celulares remover os grupos acetato, eo composto é oxidado. O número de células, medido por citometria de fluxo, que são encontrados para ser fluorescentes verde é uma indicação do número de células que estão em um estado oxidativo. CM-H 2 DCFDA é suscetível à oxidação por um grande número de ROS diferente. Esta falta de especificidade, considerando que ROS pode oxidar CM-H 2 DCFDA, faz deste composto um regente valiosa para uso nas fases iniciais de uma investigação patogênese, como este teste pode ser usado para a tela para um ambiente oxidativo celular, independentemente do que o oxigênio radical ou combinação de ROS são responsáveis ​​pelas condições celular. Uma vez que foi estabelecido que ROS estão presentes por oxidação de DCFDA 2 CM-H, em seguida, experimentos adicionais podem ser realizados para determinar qual ROS ou combinação de EROs estão envolvidas no processo de patogênese particular. Os resultados deste estudo demonstram que com a adição de peróxido de hidrogênio em um aumento DCFDA 2 CM-H fluorescência foi detectada em relação aos controles salina, indicando que este teste é um teste útil para a detecção de um ambiente oxidativo dentro G355-5 células, uma linhagem celular felina astrócitos.

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

Um método de triagem para detectar ambientes oxidativo celular é medir a oxidação do CM-H2DCFDA. Uma vez oxidada dentro de uma célula, CM-H2DCFDA muda de não-fluorescentes em um composto fluorescente. Esta mudança de fluorescência é medida por citometria de fluxo e indica o número de células em um ambiente oxidativo.

Ensaio de triagem para Estresse Oxidativo em uma linhagem de células Feline astrocitária, G355-5

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of ...

screening-assay-for-oxidative-stress-feline-astrocyte-cell-line-g355

Research

JoVE Journal

Neuroscience

14.3K visualizações.  Western University of Health Sciences. Um método de triagem para detectar ambientes oxidativo celular é medir a oxidação do CM-H2DCFDA. Uma vez oxidada dentro de uma célula, CM-H2DCFDA muda de não-fluorescentes em um composto fluorescente. Esta mudança de fluorescência é medida por citometria de fluxo e indica o número de células em um ambiente oxidativo.

Vídeo: 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.

O Ensaio neuroblasto: um ensaio para a Geração e Enriquecimento de células progenitoras neuronais de Diferenciando Progeny Neural Stem Cell utilizando citometria de fluxo

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 ...

Neurociência

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

Um ensaio de migração celular quantitativo para murinos progenitores neurais entéricas

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

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.

Analyzer celular baseado em impedância Real-Time como uma ferramenta para delinear vias moleculares envolvidas na neurotoxicidade e neuroproteção em uma linha de células neuronais

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.

A indireta Neuron-astrócitos Coculture Ensaio: An<em&gt; In Vitro</em&gt; Set-up para a investigação detalhada da Relação Neuron-glia

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.

Provocando estresse celular e morte utilizando convencional UV laser microscopia confocal

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.

Desenvolvimento de ensaio para quantificação de conteúdo elevado de Sod1 proteína mutante formação agregada em células vivas

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.

Um romance imagens Live In Vitro e fagocitose mediada por ensaio de Astrocyte usando pH sinaptossomas conjugada com indicador

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.

In Vitro Ensaio para estudar a agregação da proteína Tau e rastreio de drogas

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.

Na imagem latente de cálcio Vivo de células de cabelo linha Lateral no Zebrafish Larval

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.

Transplante Sub-Retiniano de Tecido Retiniano Derivado de Células-Tronco Embrionárias Humanas em Modelo Animal de Grande Porte Felino

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