Somos gratos aos Drs. Karl-Heinz Krause e Vincent Jaquet para ajuda e conselhos para configurar estes protocolos, e também agradecer Christoph Bauer e Jérôme Bosset da Plataforma Bioimaging do NCCR e Dr. Navin Gopaldass por seu apoio técnico. A pesquisa é apoiada por uma bolsa ProDoc da National Science Foundation suíço.

1. Visualização e medição qualitativa de ROS Produção em Fagossomos As contas OBG revestidos deve ser preparado com antecedência. Os processos de revestimento miçangas e técnica agar são adaptados a partir de referências publicadas 5,14. <ol><li> Adicionar 1 ml de suspensão de 3,0 mM esferas de sílica carboxiladas (cerca de 1,8 x 10 9 esferas) em um tubo de 1,5 ml, lavar as contas 3x com 1 ml de PBS por centrifugação rápida e centrifugação. </li><li> Ressuspender as pérolas em 1 ml de PBS contendo 25 mg / ml de cianamida (para activar as esferas de sílica carboxiladas para ligar covalentemente prelabeled BSA), incubar numa roda durante 15 min. </li><li> Lavar 3x com 1 ml de tampão de acoplamento (borato de sódio 0,1 M, pH 8,0) por centrifugação rápida (centrífuga à velocidade máxima de um tampo de mesa mini-centrifuga-se durante 5 segundos, ou, alternativamente, centrifugar a 2.000 g durante 1 min numa centrífuga de bancada) e vórtex para remover o excesso de cianamida. </li><li> Misturar os grânulos foram lavados com 500 mL de coupling tampão contendo 1 mg de verde OxyBurst (OBG) H2HFF (di-hidro-2 &#39;, 4,5,6,7,7&#39;-hexafluorofluorescein)-BSA, encher o tubo com gás azoto a partir de um padrão de gás pode antes de nivelamento, e incubar numa roda, durante 14 horas (durante a noite) à temperatura ambiente no escuro. </li><li> Lave as contas 2x com 1 ml de tampão de têmpera (250 mM de glicina em PBS) para remover não reagiu OBG, e duas vezes com tampão de acoplamento para remover o tampão de têmpera por centrifugação rápida e centrifugação. </li><li> Adicionar 1 ml de tampão de ligação contendo 50 ng de Alexa Fluor 594 éster de succinimidilo para conjugar a BSA. Encher o tubo com azoto antes de nivelamento, e incubar em uma roda durante 1,5 horas à temperatura ambiente no escuro. </li><li> Lave as contas 3x com 1 ml de tampão de têmpera por centrifugação rápida e vortex para parar a reacção, em seguida, lavar as contas 3x com 1 ml de PBS por centrifugação rápida e centrifugação. </li><li> Finalmente, voltar a suspender as pérolas em 1 ml de PBS com 2 mL de 10% w / v de azida para o armazenamento a longo prazo. Medir a concentration de grânulos em suspensão com um hemocitómetro (geralmente cerca de 1-2 x 10 9 contas / ml), encher o tubo com azoto antes de nivelamento, e armazenar a 4 ° C no escuro. </li><li> A eficiência do revestimento da fluoresceína OBG pode ser testada, verificando o espectro de emissão com excitação a 500 nm. Quando oxidado por H 2 O 2 na presença de peroxidase de rábano (HRP), a intensidade de fluorescência de grânulos oxidadas, no pico de emissão (538 nm), é de 11-12x maior que a dos grânulos revestidos não oxidados. </li><li> Colheita crescimento exponencial células Dictyostelium de um prato 10 centímetros Petri, placa diferentes densidades de células em três centímetros pratos com um plástico opticamente transparente ou com fundo de vidro, e cultivá-las durante a noite. Escolha os pratos que são cerca de 80% confluentes para o experimento. Um protocolo detalhado para o cultivo de células de Dictyostelium foi publicada em 15 de. </li><li> Substituir o meio de cultura com meio loflo (médio LF), incubar2 horas antes da experiência, a fim de diminuir a autofluorescência extracelular e intraendosomal do meio de cultura HL5C. </li><li> Em paralelo, o material fundido 10 ml de 1,5% de agar bacto em meio LF, verter o agar sobre uma superfície plana (isto é, uma placa de vidro de 10 cm x 10 cm), a fim de formar uma camada de agar de cerca de 1 mm de espessura, para esperar 10-15 min a solidificar. Cortar a camada de agar em 2 cm x 2 centímetros quadrados e coloque no meio LF para uso posterior. </li><li> Enquanto isso, prepare a fiação de disco ou grande microscópio de campo, definir a temperatura da câmara ambiental a 22 ° C e ajustar as configurações para este experimento. (Veja os comentários adicionais na discussão). </li><li> Após 2 horas de incubação, aspirar o meio LF a partir dos 3 cm de disco, mas a monocamada de células deve ainda ser coberto por uma fina película de meio. Dilui-se os grânulos revestidos a 1,5 x 10 7 esferas / ml, e adicionar 10 ul para a camada de células. </li><li> Pegue uma folha de agar quadrado, drenar o excesso de líquido, mas manter molhado. Com cuidado, coloque ªe agar quadrados no topo da camada de células. Não mova o quadrado agar quando se está deitado sobre as células. O agar é utilizado para aumentar o contato entre esferas e células, melhorando assim a absorção, e também ligeiramente comprime as células, mantendo-as melhor no plano focal da objetiva. </li><li> Coloque a tampa sobre o prato, coloque-o no palco microscópio e, automaticamente, tirar fotos nos canais vermelho, verde e fase a cada 1 min de 2 horas ou mais. </li><li> Selecione e focar em eventos celulares que contêm todo o processo de fagocitose. Mesclar os 3 canais otimizados e montar as imagens em um filme usando o software de processamento de imagem profissional. Quantificar intensidades de fluorescência de cada contas selecionadas em canais vermelho e verde, ea proporção de verde / vermelho vai refletir a dinâmica phagosomal produção ROS das células. </li></ol> 2. Medição quantitativa e Média taxa de transferência de superóxido intracelular Produção <ol><li> Coletar one 80% confluentes prato de Dictyostelium, em 10 ml de meio HL5C. Células Centrífuga Dictyostelium a 850 xg durante 5 min, com cuidado e o excesso de meio completamente aspirado. Ressuspender as células em tampão SS6.4 [0,12 M de sorbitol em tampão de Sorensen (14,69 mM de KH 2 PO 4, e 6,27 mM de Na 2 HPO 4), pH 6,4], contagem de células e diluir para uma densidade final de 6 x 10 6 células / ml (ver comentários adicionais na discussão). </li><li> Adicionar 50 ul de suspensão celular em cada poço de uma branca não transparente placa de 96 poços (ver comentários adicionais na discussão). </li><li> Diluir estoque DHE (30 mM em DMSO) a 500 vezes com SS6.4, pipeta 50 ul de DHE diluída para cada poço utilizando uma pipeta de canais múltiplos, se necessário. A concentração final da DHE é de 30 uM. A reacção inicia-se imediatamente. </li><li> Estímulos ou inibidores podem ser adicionados neste momento, ou em outros pontos de tempo de acordo com as necessidades específicas. </li><li> Use o ponto de &quot;leitura &amp; # top final34, Modo de um leitor de microplacas de fluorescência, fluorescência com excitação / emissão a 522 nm/605 nm, ler todos os 2 minutos durante 1 hora, a vibração da média de 5 segundos / min, a 22 ° C. </li></ol> 3. Quantitativa e High Throughput Medição de Extracelular H 2 O 2 Produção <ol><li> Siga os mesmos passos descritos em 2.1 e 2.2. </li><li> Prepare HRP diluída pela adição de 5 ul de estoque de HRP (100 U / ml em água destilada) em 10 ml de SS6.4, vórtice ou inverter o tubo para misturar bem e manter em gelo para posterior utilização. A solução HRP diluída é de 0,05 U / ml. </li><li> Preparar a mistura de reacção por mistura de AUR o estoque AUR (AUR 10 mM em DMSO) e a solução diluída em tampão HRP SS6.4 para as concentrações finais de 6,25 jiM, e 0,005 U / ml, respectivamente. Como um exemplo, para preparar a mistura de reacção para 40 reacções, misturar 1,600 mL de SS6.4 com 400 ul de HRP e diluída com 2,5 ml de solução de estoque AUR. </li><li> Adicionar 50 ul de mistura AURtura em cada poço utilizando uma pipeta de canais múltiplos, se necessário. Agora, a reacção começa. </li><li> Estímulos ou inibidores podem ser adicionados neste momento, ou em outros pontos de tempo de acordo com as necessidades específicas. </li><li> Use o ponto de modo final &quot;top leitura&quot; de uma micro leitor de placas de fluorescência, com fluorescência excitação / emissão a 530 nm nm/590, leia cada 2 minutos para 1 hora, a vibração da média 5 seg / min, a 22 ° C. </li></ol>

<ol>
	<li>Bedard, K., Krause, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NOX+family+of+ROS-generating+NADPH+oxidases:+physiology+and+pathophysiology.">The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology.</a> Physiol. Rev. 87, 245-313 (2007).</li><li>West, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR+signalling+augments+macrophage+bactericidal+activity+through+mitochondrial+ROS.">TLR signalling augments macrophage bactericidal activity through mitochondrial ROS.</a> Nature. 472, 476-480 (2011).</li><li>Fey, P., Gaudet, P., Pilcher, K. E., Franke, J., Chisholm, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dictyBase+and+the+Dicty+Stock+Center.">dictyBase and the Dicty Stock Center.</a> Methods Mol. Biol. 346, 51-74 (2006).</li><li>Basu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dictyBase+2013:+integrating+multiple+Dictyostelid+species.">dictyBase 2013: integrating multiple Dictyostelid species.</a> Nucleic Acids Res. 41, 676-683 (2013).</li><li>VanderVen, B. C., Yates, R. M., Russell, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraphagosomal+measurement+of+the+magnitude+and+duration+of+the+oxidative+burst.">Intraphagosomal measurement of the magnitude and duration of the oxidative burst.</a> Traffic. 10, 372-378 (2009).</li><li>Cohn, C. A., Simon, S. R., Schoonen, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+fluorescence-based+techniques+for+the+quantification+of+particle-induced+hydroxyl+radicals.">Comparison of fluorescence-based techniques for the quantification of particle-induced hydroxyl radicals.</a> Part. Fibre Toxicol. 5, 2 (2008).</li><li>Snyrychova, I., Ayaydin, F., Hideg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+hydrogen+peroxide+in+leaves+in+vivo+-+a+comparison+of+methods.">Detecting hydrogen peroxide in leaves in vivo - a comparison of methods.</a> Physiol. Plant. 135, 1-18 (2009).</li><li>Rodrigues, J. V., Gomes, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+superoxide+and+hydrogen+peroxide+detection+in+biological+assays.">Enhanced superoxide and hydrogen peroxide detection in biological assays.</a> Free Radic. Biol. Med. 49, 61-66 (2010).</li><li>Chen, J., Rogers, S. C., Kavdia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Kinetics+of+Dihydroethidium+Fluorescence+with+Superoxide+Using+Xanthine+Oxidase+and+Hypoxanthine+Assay.">Analysis of Kinetics of Dihydroethidium Fluorescence with Superoxide Using Xanthine Oxidase and Hypoxanthine Assay.</a> Ann. Biomed. Eng. , (2012).</li><li>Lee, C. W., Chen, Y. C., Ostafin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+accuracy+of+Amplex+Red+assay+for+hydrogen+peroxide+in+the+presence+of+nanoparticles.">The accuracy of Amplex Red assay for hydrogen peroxide in the presence of nanoparticles.</a> J. Biomed. Nanotechnol. 5, 477-485 (2009).</li><li>Guimaraes-Ferreira, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+creatine+supplementation+decreases+reactive+oxygen+species+content+with+no+changes+in+expression+and+activity+of+antioxidant+enzymes+in+skeletal+muscle.">Short-term creatine supplementation decreases reactive oxygen species content with no changes in expression and activity of antioxidant enzymes in skeletal muscle.</a> Eur. J. Appl. Physiol. 112, 3905-3911 (2012).</li><li>Peng, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+peroxidase+7+protects+against+oxidative+DNA+damage+in+oesophageal+cells.">Glutathione peroxidase 7 protects against oxidative DNA damage in oesophageal cells.</a> Gut. 61, 1250-1260 (2012).</li><li>Walk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide+enhances+bactericidal+activity+in+Dictyostelium+discoideum+cells.">Lipopolysaccharide enhances bactericidal activity in Dictyostelium discoideum cells.</a> Dev. Comp. Immunol. 35, 850-856 (2011).</li><li>Fukui, Y., Yumura, S., Yumura, 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=Agar-overlay+immunofluorescence:+high-resolution+studies+of+cytoskeletal+components+and+their+changes+during+chemotaxis.">Agar-overlay immunofluorescence: high-resolution studies of cytoskeletal components and their changes during chemotaxis.</a> Methods Cell Biol. 28, 347-356 (1987).</li><li>Fey, P., Kowal, A. S., Gaudet, P., Pilcher, K. E., Chisholm, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+growth+and+development+of+Dictyostelium+discoideum.">Protocols for growth and development of Dictyostelium discoideum.</a> Nat. Protoc. 2, 1307-1316 (2007).</li><li>Bloomfield, G., Pears, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+signalling+required+for+multicellular+development+of+Dictyostelium.">Superoxide signalling required for multicellular development of Dictyostelium.</a> J. Cell. Sci. 116, 3387-3397 (2003).</li><li>Dieckmann, R., Gopaldass, N., Escalera, C., Soldati, T., Deretic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagosomes+and+Phagosomes.">Autophagosomes and Phagosomes.</a> Methods in Molecular Biology. 445, 317-328 (2008).</li><li>Amir, Y., Edward, O. -. A., Utpal, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+in+vivo+detection+of+reactive+oxygen+species.">A protocol for in vivo detection of reactive oxygen species.</a> Proto. Exch. , (2008).</li><li>Neuhaus, E. M., Soldati, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+myosin+I+is+involved+in+membrane+recycling+from+early+endosomes.">A myosin I is involved in membrane recycling from early endosomes.</a> J. Cell Biol. 150, 1013-1026 (2000).</li></ol>

Não há conflitos de interesse declarados.

Em comparação com os métodos anteriormente descritos, o ensaio de OBG visualiza dinamicamente o processo de geração de ROS phagosomal ao nível da célula individual, em vez de medir o sinal médio ROS a partir de uma população de células. Tais métodos população de amostragem tendem a obscurecer a informação crítica causada por fagocitose não-sincrónico. Nós adaptado com sucesso DHE e AUR ensaios para Dictyostelium e otimizados os protocolos. Mais importante, nós especificados os tipos e local...

Espécies reativas de oxigênio (ROS) estão envolvidos em uma ampla variedade de processos biológicos, tais como a defesa do hospedeiro, a sinalização, o desenvolvimento do tecido e resposta à lesão, bem como hipertensão e câncer. O phagosomal NADPH oxidase máquinas bem estudado é dedicado a geração de ROS rápida, conhecida como a explosão oxidativa, para matar as bactérias ingeridas em phagosomes dos neutrófilos 1. Além disso, o vazamento de elétrons como um subproduto da cadeia respiratória mitocondrial foi previamente pensado para ser responsável apenas por uma fonte não regulamentada de ROS. Mas, recentemente, foi identificado como um mecanismo importante para contribuir para a matança intraphagosomal de bactérias nos macrófagos do rato 2. Nos últimos anos, a ameba social Dictyostelium, tornou-se um modelo poderoso e popular para o estudo de células mecanismos intrínsecos da resposta imune inata. De fato, Dictyostelium e fagócitos humanos partilham uma surpreendentemente alto nível de conservação em molecular máquinas responsáveis ​​por bactérias sentindo, imersão e matando 3,4. Os homólogos de proteínas e enzimas relacionadas com a produção de ROS e toxinas, tais como oxidase de NADPH, catalases, superóxido dismutase, pode ser encontrado em humanos e Dictyostelium. À medida que a ameba melhor estudada, Dictyostelium apresenta várias vantagens únicas sobre sistema modelo de mamíferos. Eles crescer à temperatura ambiente, sem a necessidade de CO 2, com um tempo de duplicação de 8-10 horas. Eles podem ser facilmente mantidas como culturas aderentes ou em suspensão. Além disso, graças ao seu genoma haplóide completamente seqüenciado e anotado, e para a manipulação genética fácil, Dictyostelium tornou-se um modelo muito atraente organismo experimental. Em estudos anteriores, vários derivados clorados e fluorados de fluoresceína (conhecidos coletivamente como OxyBurst verde, OBG) que emitem fluorescência após a oxidação por ROS, têm sido usados ​​como um repórter ROS. Cell-permeante esterified derivados são usados ​​para medir citoplasmática ROS, enquanto dextrano ou derivados acoplados à proteína de células-impermeabilizante são usados ​​para medir ROS extracelular. Em particular, grânulos de OBG-ASB revestidas já tiverem sido usadas para detectar a produção de ROS phagosomal em células de mamíferos 5. No entanto, a abordagem baseada em leitor de microplacas só pode dar uma curva de geração de ROS em média de uma população de células. Com o presente protocolo, usando Dictyostelium, um dos fagócitos profissionais e condições experimentais otimizadas, obtemos fagocitose estável e eficiente, sem conjugar qualquer opsonina sobre as contas. DHE tem sido usada em vários sistemas de modelos, tais como os neutrófilos e macrófagos de mamífero, para detectar a produção de ROS 6-9. Enquanto isso, existem algumas controvérsias sobre a especificidade e sensibilidade do método 8,10. Como uma versão melhorada de Amplex Red, a intensidade de fluorescência de AUR é menos sensível ao pH, o que torna mais adequado para medirROS em ambientes não-neutras ou fracamente ácidas. AUR foi recentemente aplicado em vários sistemas de mamíferos 11,12, mas a sua utilização em modelos não mamíferos, o que muitas vezes necessitam de meios de crescimento ligeiramente ácido, não tem sido relatado ainda. Além disso, não existe um protocolo publicado quantitativamente e dinamicamente medir a produção e localização ROS na ameba Dictyostelium sociais. O objetivo do conjunto de protocolos apresentados é fornecer uma solução fácil e versátil para monitorar vários ROS e sua localização, e ainda dar insights sobre os mecanismos celulares relacionados com o ROS. Para o ensaio de OBG, usamos a microscopia ao vivo para acompanhar todo o processo de geração de ROS phagosomal após a captação de contas OBG revestidos por células Dictyostelium, e forneceu uma nova abordagem para estudar o mecanismo de matar intraphagosomal de bactérias. Nós otimizamos de médio rendimento DHE e AUR ensaios em Dictyostelium, para medir Superox intracelularide e H 2 O 2 extracelular produção, respectivamente, utilizando LPS como um potente estimulador de ROS. Observe que o LPS foi recentemente demonstrado que o aumento da actividade bactericida de Dictyostelium direcção bactérias ingeridas 13. Além disso, o tratamento com DEDTC e catalase confirmado explicitamente que estes dois métodos de medir especificamente diferentes tipos e localizações subcelulares de ROS em Dictyostelium. Finalmente, o ensaio de OBG pode ser adaptado a qualquer célula fagocítica aderente, por opsonizantes ainda mais os grânulos com ligandos para receptores de células fagocíticas animais. Em princípio, os ensaios de DHE e AUR pode ser também para medições em qualquer célula aderente ou não aderente em condições experimentais apropriadas aperfeiçoá-lo.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>OxyBURST Green H2HFF BSA</td>
			<td>Invitrogen</td>
			<td>O-13291</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594</td>
			<td>Invitrogen</td>
			<td>A-20004</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxylated silica beads</td>
			<td>Kisker Biotech</td>
			<td>PSi-3.0COOH</td>
			<td></td>
		</tr>
		<tr>
			<td>HL5C medium</td>
			<td>ForMedium</td>
			<td>HLC0102</td>
			<td></td>
		</tr>
		<tr>
			<td>LoFlo medium</td>
			<td>ForMedium</td>
			<td>LF1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto agar</td>
			<td>BD</td>
			<td>204010</td>
			<td></td>
		</tr>
		<tr>
			<td>Dihydroethidium</td>
			<td>Sigma</td>
			<td>37291-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex UltraRed</td>
			<td>Invitrogen</td>
			<td>A36006</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase</td>
			<td>Roche</td>
			<td>10108090001</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyldithiocarbamate (DEDTC)</td>
			<td>Sigma</td>
			<td>D3506-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma</td>
			<td>C9322-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanamide</td>
			<td>Sigma</td>
			<td>187364-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharides</td>
			<td>Sigma</td>
			<td>L2630-25G</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>ibidi &#956;-Dish 35 mm, high</td>
			<td>ibidi</td>
			<td>81156</td>
			<td>Bottom made of optically clear plastic</td>
		</tr>
		<tr>
			<td>MatTek dish 35 mm</td>
			<td>MatTek corporation</td>
			<td>P35G-1.5-14-C</td>
			<td>Bottom made of a glass coverslip</td>
		</tr>
		<tr>
			<td>Scepter Cell Counter</td>
			<td>Merck Millipore</td>
			<td>PHCC00000</td>
			<td></td>
		</tr>
		<tr>
			<td>White 96 well plate</td>
			<td>Nunc</td>
			<td>236108</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>SORVALL</td>
			<td>Legend RT</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>BioTek</td>
			<td>Synergy Mx</td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting, visualizing and quantitating the generation of reactive oxygen species in an amoeba model system

Espécies reativas de oxigênio (ROS) compreendem uma gama de moléculas reativas e de curta duração contendo oxigênio, que são dinamicamente interconvertidos ou eliminados ou cataliticamente ou espontaneamente. Devido aos curtos períodos de vida da maioria ROS ea diversidade de suas fontes e localizações subcelulares, um quadro completo só pode ser obtida por meio de medições cuidadosas usando uma combinação de protocolos. Aqui, apresentamos um conjunto de três protocolos diferentes usando OxyBurst Green (OBG) revestida de contas, ou dihydroethidium (DHE) e Amplex Ultrared (AUR), para monitorar qualitativa e quantitativamente vários ROS em fagócitos profissionais, como Dictyostelium. Nós otimizamos os processos de revestimento contas e miçangas OBG revestidos usados ​​e microscopia ao vivo para visualizar dinamicamente intraphagosomal geração ROS no nível de uma única célula. Foram identificados lipopolissacarídeo (LPS) de E. coli como um potente estimulador para a geração de ROS em Dictyostelium. Além disso, nós dtempo real eveloped, ensaios de médio rendimento, utilizando DHE e AUR para medir quantitativamente superóxido intracelular e extracelular H 2 O 2 a produção, respectivamente.

A geração de ROS em phagosomes pode ser visualizada qualitativamente e dinamicamente por microscopia (S1 do ficheiro). A fluorescência vermelha emitida pela Alexa fluor 594 é insensível pH e mantém-se constante no ambiente phagosomal, enquanto que a oxidação de OBG aumenta a fluorescência no canal verde. O espectro de emissão de ensaios in vitro e oxidado OBG grânulos revestidos não oxidados são comparados na Figura 1B, que mostra um aumento significativo ...

Watch this Scientific Journal Video about Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System at JoVE.com

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Nós adaptamos um conjunto de protocolos para a medição de espécies reativas de oxigênio (ROS) que podem ser aplicados em vários modelos celulares ameba e mamíferos para estudos qualitativos e quantitativos.

Detectando, visualizar e quantificar a geração de espécies reativas de oxigênio em um modelo de sistema Amoeba

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

detecting-visualizing-quantitating-generation-reactive-oxygen-species

Research

JoVE Journal

Biology

16.0K visualizações.  University of Geneva.  Nós adaptamos um conjunto de protocolos para a medição de espécies reativas de oxigênio (ROS) que podem ser aplicados em vários modelos celulares ameba e mamíferos para estudos qualitativos e quantitativos. 

Vídeo: Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

Visualizar o coração batendo no Drosophila</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Biologia

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

Ressonância paramagnética eletrônica Micro-imagem das Espécies ao vivo para o Mapeamento de oxigênio

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself Dispositivo de Recuperação de amostras criopreservadas cair acidentalmente em Tanques de armazenamento criogênico

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

Geração, purificação, caracterização e de Cell-invasivos espécies de proteína Disc1

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of 'escape' of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.

Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

Geração de mutantes sem marcadores marcados e no modelo de cianobactérias da espécie

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

Roedor Trabalho Modelo do coração para o estudo do desempenho do miocárdio eo consumo de oxigênio

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Visualizing the Early Stages of Phagocytosis

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system &#8212; such as neutrophils, dendritic cells, and macrophages &#8212; retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4.
Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 &#181;m, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

Visualizando os estágios iniciais da fagocitose

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Estabelecimento de um pH extracelular ácido sistema de cultura

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient production of ROS in situ efficiently activates cell proliferation in stem cell niches and stimulates tissue regeneration as strongly manifested through the acceleration of burn healing and hair follicle growth processes. The protocol is based on a regulatable photodynamic treatment that treats the tissue with precursors of the endogenous photosensitizer protoporphyrin IX and further irradiates the tissue with red light under tightly controlled physicochemical parameters. Overall, this protocol constitutes an interesting experimental tool to analyze ROS biology.

The aim of this protocol is to induce transient in vivo production of nonlethal levels of reactive oxygen species (ROS) in mouse skin, further promoting physiological responses in the tissue.

Estimulação de nichos de células-tronco e regeneração tecidual em pele de camundongo por fotogeração dependente de protoporfirina IX comutável de espécies reativas de oxigênio in situ

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient ...