Este trabalho foi financiado pelo National Institutes of Health (CA142664).

1. Detecção de ROS usando carboxi-H 2 DCFDA Carboxi-H 2 DCFDA não é fluorescente, mas na presença de ROS, quando este reagente é oxidado, torna-se verde fluorescente. <ol><li> Imediatamente antes da utilização, prepare uma solução estoque fresca de DCFDA 2 carboxi-H em dimetilsulfóxido estéril (DMSO) ou etanol 100%. Evite descongelar múltiplos / congelar os ciclos de sua tintura. </li><li> Lavar as células com tampão HEPES solução de sal (HBSS) ou tampão fosfato (PBS) para remover os vestígios do meio original. </li><li> Carregar as células com o corante (dye eo controle, cf seção de nota). Neste ensaio usamos Jurkat, uma linha de células humanas de leucemia. Use carboxi-H 2 DCFDA em uma concentração final de 1 Hm em meio de cultura com soro normal reduzida (2%). </li><li> Incubar as culturas por 30 minutos no escuro, em uma incubadora convencional (37 ° C, 5% de CO2). Descartar todas as soluções de corante não utilizados. </li><li> Remover carboxi-H 2 DCFDA contendo meio e lavar duas vezes com HBSS ou PBS. A partir deste passo em frente, proteger as células da luz. </li><li> Adicione meio fresco contendo sua droga de escolha para o carboxi-H 2 DCFDA células-carregados e incubar conforme desejado. Para este exemplo, usamos H 2 O 2 (0,03%) por 1 hora. </li><li> Avaliar ROS por analisar imediatamente suas células por citometria de fluxo através do canal de FL1 (fluorescência verde), ou pelo leitor de placas de fluorescência, ou por microscopia de fluorescência. A fluorescência pode ser detectada por meio de excitação e emissão comprimentos de onda que são apropriadas para fluorescência verde. </li></ol> Nota: Os controlos devem incluir carboxi-H 2 DCFDA-carregado células não tratadas e imaculada células não tratadas. Carboxi-H 2 DCFDA é conhecido por detectar peróxidos mas também pode ser oxidado pelo ROS outros. Este reagente também pode ser modificado por outros meios, a oxidação de corante controle insensível como a 5 - (e-6)-carboxi-2 &#39;, 7&#39;-dichlorofluorescein diacetato (carboxi-DCFDA) deve ser incluído no teste. 2. Medição da produção de óxido nítrico (NO) Você vai precisar de sulfanilamida e N-1-napthylethylenediamine dicloridrato (NED) soluções e padrão de nitrito. Este ensaio é chamado de ensaio de Griess. NED solução: faça uma solução de 0,1% do N-1-napthylethylenediamine dicloridrato diluída em solução estéril water.Sulfanilamide: Faça uma solução de 1% sulfanilamida diluídos em ácido fosfórico 5%. Padrão de nitrito: Diluir o padrão de nitrito de sódio 0,1 M de ações para 100μM em meio estéril, fazer uma diluição em série no mesmo meio. Condições de armazenamento: Loja de produtos químicos, conforme indicado pelo fabricante à temperatura ambiente. Quando reconstituído, NED e soluções Sulfanilamida são armazenados imediatamente após o uso, a 4 ° C, no escuro, e por um máximo de 3 meses. <ol><li> Cultura de células em um prato de 96 poços,use triplicatas para cada condição, e incluem controles adequados de acordo com suas experiências. </li><li> Tratar células para induzir a produção de NO. Em nosso experimento utilizamos lipopolissacarídeo (LPS) (100 ng / ml) e IL-4 recombinante para o tratamento de nossas células. Neste protocolo, foram utilizados 264,7 RAW, um rato linha de células de macrófagos (Mouse linhagem de células leucêmicas monócitos macrófagos). </li><li> No dia do ensaio, colocar ambos os reagentes à temperatura ambiente. </li><li> Girar seu prato, colher sobrenadantes de células e transferência 50μl para uma placa de 96 novas também. Prepare limitando poços de diluição da solução estoque padrão para fazer uma curva padrão. </li><li> Adicionar 50μl de solução de sulfanilamida a cada amostra e controle bem e misture bem. </li><li> Incubar à temperatura ambiente por 10 minutos no escuro. </li><li> Adicionar 50μl de N-1 napthylethylenediamine solução de dicloridrato de cada amostra e controle bem e misture bem. </li><li> Incubar à temperatura ambiente por 10 minutos no escuro. </li><li> Medida absorbance imediatamente, usando um leitor de placas com filtro de comprimentos de onda entre 520nm e 550nm. </li></ol> Se você utilizar diferentes placa / prato tamanhos, use 1/1/1 volume para cada solução e sobrenadante da amostra. A cor violeta vai aparecer nos poços positivos. Resultados obtidos com o padrão irá ajudá-lo a verificar a estabilidade de suas soluções. 3. Detecção de ROS ação: proteínas oxidadas Um método diferente para identificar a produção de ROS é olhar para os resultados finais, detectando a oxidação de proteínas. De fato, ROS modificar glutationa, um antioxidante que é expresso na maioria das células e desempenha um papel protetor contra ROS. Após a oxidação por ROS, modificação da glutationa reduzida (GSH) os resultados do grupo (tiol) sulfidrila de sua cisteína estar ligada a uma segunda glutationa através de uma ponte dissulfeto. Isso leva à formação de uma proteína dimerizada (GSSG proteína oxidada). GSH pode ser restaurada através de modificação de GSSG pela glutationa reductase da enzima. O aumento da relação GSSG / GSH reflete o estresse oxidativo. O ensaio a seguir é baseado na detecção e quantificação destas proteínas oxidadas. Este método não é seletivo para ROS específicos, mas sim detectar os efeitos do NO, H 2 O 2, O 2 - e outras ROS. Aqui medimos a quantidade total de oxidada (GSSG) e reduzida (GSH) a glutationa usando sinais bioluminescente. <ol><li> Semente de suas células em uma placa de 96 poços (branco / transparente, fundo plano), como de costume. Para nosso experimento, foi utilizado 1x10 4 células por poço de aderentes Raw 264,7 e A549 e células Jurkat suspensão. Dependendo do tamanho de suas células, estes números podem ser ajustados. Recomendamos para as células placa aderente um dia antes do teste para permitir que eles atribuem ao prato. Suficiente poços devem estar preparados para a detecção da proteína oxidada e reduzida, bem como para &quot;apenas médio&quot; ecélulas não tratadas como controle. Use triplicatas para todas as condições. </li><li> Tratar as células com o seu medicamento para o tempo necessário. Aqui nós tratamos as células Jurkat e células A549 por 1h com H 2 O 2 (a 5mM e 2,5 mm, respectivamente). Raw 264,7 células foram tratadas com 200ng/ml de LPS por 16 h. Durante este tempo de incubação, trazer todos os seus reagentes à temperatura ambiente (RT). Faça todos os reagentes não mais que 30 minutos antes de realizar o teste. A tabela abaixo mostra os volumes de reagentes por poço. Quando possível, é importante para remover a mídia que contém o agente redutor antes de prosseguir com o teste. </li></ol><table border="1"><tbody><tr><td rowspan="2"></td><td colspan="2"> Células aderentes </td><td colspan="2"> Células em suspensão </td></tr><tr><td> Lise Glu totais </td><td> Oxidado Lise Glu </td><td> Lise Glu totais </td><td> Oxidado Lise Glu </strong&gt; </td></tr><tr><td> NEM, 25mm </td><td> nenhum </td><td> 0.5μl </td><td> nenhum </td><td> 0.5μl </td></tr><tr><td> Luciferina-NT </td><td> 1 ml </td><td> 1 ml </td><td> 1 ml </td><td> 1 ml </td></tr><tr><td> Tampão de Lise passiva, 5X </td><td> 10μl </td><td> 10μl </td><td> 10μl </td><td> 10μl </td></tr><tr><td> Água destilada </td><td> 39.0μl </td><td> 38.5μl </td><td> 14μl </td><td> 13.5μl </td></tr><tr><td> Volume final por poço </td><td> 50μl </td><td> 50μl </td><td> 25μl </td><td> 25μl </td></tr></tbody></table><ol start="3"><li> Remova o meio de poços com células aderentes. Não remova o meio de poços com células em suspensão. </li><li> Adicionar reagente de lise glutationa reduzida ou reagente de lise glutationa oxidada correspondentes aos poços e agitar por 5 minutos em temperatura ambiente. </li><li> Adicionar o reagente geração luciferina e incubate por 30 minutos em temperatura ambiente. </li><li> Adicione o reagente de detecção Luciferina e incubar por 15 minutos em temperatura ambiente. </li><li> Leia o sinal bioluminescente no tempo de integração de 0,25-1 segundo por bem utilizar um leitor de placas luminômetro. </li></ol><table border="1"><tbody><tr><td></td><td> Células aderentes </td><td> Células em suspensão </td></tr><tr><td> Número de células por poço </td><td> 1x10 4 </td><td> 1x10 4 </td></tr><tr><td> Suspensão celular por drogas + bem </td><td> 100 l, para ser removido antes de 3,4 </td><td> 25μl, para não ser removido </td></tr><tr><td> Reagente de Lise glutationa reduzida </td><td> 50 ul </td><td> 25μl </td></tr><tr><td> Reagente de Lise Glutationa oxidada </td><td> 50 ul </td><td> 25μl </td></tr><tr><td> Luciferina Reagente Generation </td><td> 50 ul </td><td> 50 ul </td></tr><tr><td> Luciferina Reagente de Detecção </td><td> 100 l </td><td> 100 l </td></tr></tbody></table> 4. Resultados representativos: <img alt="Figura 1" src="/files/ftp_upload/3357/3357fig1.jpg" /> Detecção figura 1 do ROS usando carboxi-H2DCFDA corante. Células Jurkat (linhagem de células humanas de leucemia) tratados com H 2 O 2 foram comparados com os não tratados células. ROS induz a modificação de DCFDA 2 carboxi-H que fluoresce verde como detectada por citometria de fluxo, o pico fluorescentes em H 2 O 2 células tratadas mudança em comparação com os picos nos controles (H 2 O 2 células tratadas marcadas com corante de oxidação insensível e não tratados com células coradas com DCFDA 2 carboxi-H). Resultados confirmam a presença de ROS em células tratadas. <img alt="Figura 2" src="/files/ftp_upload/3357/3357fig2.jpg" /> Figura 2 Detecção de of NO utilizando reagentes Griess. RAW 264,7 células (mouse macrófagos) foram tratados com LPS e IL-4. Um aumento significativo na produção de NO foi detectada em células tratadas em relação ao controle de células não tratadas. <table border="1"><tbody><tr><td> Linhagens de células </td><td> Não tratados </td><td> Tratado </td></tr><tr><td> Raw 264,7 </td><td> 13,0 </td><td> 8,3 </td></tr><tr><td> A549 </td><td> 21,6 </td><td> 10,5 </td></tr><tr><td> Jurkat </td><td> 5,2 </td><td> 2,8 </td></tr></tbody></table> Detecção tabela 1 de ROS mediada oxidação de proteínas. RAW 264,7 células foram tratadas com LPS, Jurkat e A549 (câncer de pulmão humano), as células foram tratadas com H 2 O 2. Os resultados são expressos como a razão reduzida (GSH) / oxidada (GSSG) glutationa. Rácios mais baixos de glutationa (GSH) / (GSSG) foram detectados em células tratadas em comparação comcontrole de células não tratadas, revelando que as proteínas foram mais oxidado em células tratadas.

<ol>
	<li>Guzik, T. J., Korbut, R., Adamek-Guzik, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+and+superoxide+in+inflammation+and+immune+regulation.">Nitric oxide and superoxide in inflammation and immune regulation.</a> J Physiol Pharmacol. 54, 469-487 (2003).</li><li>Perl, A., Gergely, P., Banki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+T+cells+of+patients+with+systemic+lupus+erythematosus.">Mitochondrial dysfunction in T cells of patients with systemic lupus erythematosus.</a> Int Rev Immunol. 23, 293-313 (2004).</li><li>Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T., Mazur, M., Telser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+and+antioxidants+in+normal+physiological+functions+and+human+disease.">Free radicals and antioxidants in normal physiological functions and human disease.</a> Int J Biochem Cell Biol. 39, 44-84 (2007).</li><li>Droge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+in+the+physiological+control+of+cell+function.">Free radicals in the physiological control of cell function.</a> Physiol Rev. 82, 47-95 (2002).</li><li>Nakamura, K., Yube, K., Miyatake, A., Cambier, J. C., Hirashima, M. <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+CD4+D3-D4+membrane+proximal+extracellular+domain+for+the+inhibitory+effect+of+oxidative+stress+on+activation-induced+CD4+down-regulation+and+its+possible+role+for+T+cell+activation.">Involvement of CD4 D3-D4 membrane proximal extracellular domain for the inhibitory effect of oxidative stress on activation-induced CD4 down-regulation and its possible role for T cell activation.</a> Mol Immunol. 39, 909-921 (2003).</li><li>Los, M., Droge, W., Stricker, K., Baeuerle, P. A., Schulze-Osthoff, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+as+a+potent+activator+of+T+lymphocyte+functions.">Hydrogen peroxide as a potent activator of T lymphocyte functions.</a> Eur J Immunol. 25, 159-165 (1995).</li><li>Deem, T. L., Cook-Mills, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+cell+adhesion+molecule+1+(VCAM-1)+activation+of+endothelial+cell+matrix+metalloproteinases:+role+of+reactive+oxygen+species.">Vascular cell adhesion molecule 1 (VCAM-1) activation of endothelial cell matrix metalloproteinases: role of reactive oxygen species.</a> Blood. 104, 2385-2393 (2004).</li><li>Pacher, P., Beckman, J. S., Liaudet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+and+peroxynitrite+in+health+and+disease.">Nitric oxide and peroxynitrite in health and disease.</a> Physiol Rev. 87, 315-424 (2007).</li><li>Griendling, K. K., Sorescu, D., Lassegue, B., Ushio-Fukai, M. <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+protein+kinase+activity+and+gene+expression+by+reactive+oxygen+species+and+their+role+in+vascular+physiology+and+pathophysiology.">Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology.</a> Arterioscler Thromb Vasc Biol. 20, 2175-2183 (2000).</li><li>Loh, K., Deng, H., Fukushima, A., Cai, X., Boivin, B., Galic, S., Bruce, C., Shields, B. J., Skiba, B., Ooms, L. M., Stepto, N., Wu, B., Mitchell, C. A., Tonks, N. K., Watt, M. J., Febbraio, M. A., Crack, P. J., Andrikopoulos, S., Tiganis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+enhance+insulin+sensitivity.">Reactive oxygen species enhance insulin sensitivity.</a> Cell Metab. 10, 260-272 (2009).</li><li>Goldstein, B. J., Mahadev, K., Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+paradox:+insulin+action+is+facilitated+by+insulin-stimulated+reactive+oxygen+species+with+multiple+potential+signaling+targets.">Redox paradox: insulin action is facilitated by insulin-stimulated reactive oxygen species with multiple potential signaling targets.</a> Diabetes. 54, 311-321 (2005).</li></ol>

Recebemos o apoio da Promega para esta publicação.

Várias situações patológicas como doenças inflamatórias, câncer, isquemia / reperfusão, e também tratamentos como a radioterapia ou quimioterapia (cisplatina ou seja) induzem a superprodução de ROS. Assim, a detecção e medição dos níveis de ROS é importante em muitos básica, estudos pré-clínicos e clínicos. No entanto, ROS têm semi-vidas muito curtas e pode ser complicado de detectar. Aqui, propomos testes simples que são usados ​​rotineiramente e amplamente aceites para a detecção de produ?...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comments (opcional) </th></tr><tr><td> 5 - (e-6)-carboxi-2 &#39;, 7&#39;-diacetato diclorofluoresceína (carboxi-DCFDA) </td><td> Sondas moleculares </td><td> C369 </td><td> controle </td></tr><tr><td> carboxi-H 2 DCFDA </td><td> Sondas moleculares </td><td> C400 </td><td></td></tr><tr><td> Sulfanilamida </td><td> Sigma </td><td> S9251-100G </td><td></td></tr><tr><td> N-1-napthylethylenediamine dicloridrato </td><td> Sigma </td><td> N9125-10G </td><td></td></tr><tr><td> Padrão de nitrito </td><td> Sigma </td><td> 237213-100G </td><td></td></tr><tr><td> GSH / GSSG-Glo Assay </td><td> Promega </td><td> V6612 </td><td> Para quantificar oxidado, reduzido ou oxidado / glutationa reduzida </td></tr></tbody>
</table>

production and detection of reactive oxygen species (ros) in cancers

Espécies reativas de oxigênio incluir um número de moléculas que danificam o DNA e RNA e oxidar proteínas e lipídios (peroxydation lipídica). Estas moléculas reativas de oxigênio contêm um e incluem H 2 O 2 (peróxido de hidrogênio), NO (óxido nítrico), O 2 - (ânion óxido), peroxinitrito (ONOO -), ácido hydrochlorous (HOCl) e radical hidroxila (OH -) . Espécies oxidativas são produzidos não apenas em situações patológicas (câncer, reperfusão isquêmica /, patologias neurológicas e cardiovasculares, doenças infecciosas, doenças inflamatórias 1, doenças auto-imunes 2, etc ...), mas também em situações fisiológicas (não patológicas), tais como o metabolismo celular 3 , 4. De fato, ROS desempenham um papel importante em muitas vias de sinalização celular (proliferação, ativação de células 5, 6, 7 migração etc.). ROS pode ser prejudicial (então é referido como&quot;Stress oxidativo e nitrosativo&quot;) quando produzida em quantidades elevadas nos compartimentos intracelulares e células geralmente respondem a ROS por upregulating antioxidantes como a superóxido dismutase (SOD) e catalase (CAT), glutationa peroxidase (GPx) e glutationa (GSH), que protege convertendo-los perigosos radicais livres em moléculas inofensivas (água, por exemplo). Vitaminas C e E também têm sido descritos como scavengers ROS (antioxidantes). Os radicais livres são benéficos em pequenas quantidades 3. Macrófagos e neutrófilos mediada por resposta imune envolvem a produção e liberação de NO, que inibe vírus, patógenos e da proliferação do tumor 8. NO também reage com ROS outros e, assim, também tem um papel como um desintoxicante (scavenger ROS). Finalmente NO age em navios para regular o fluxo de sangue que é importante para a adaptação do músculo ao exercício prolongado 9, 10. Várias publicações têm demonstrado também que ROS estão envolvidas na sens insulinaitivity 11, 12. Vários métodos para avaliar a produção de ROS estão disponíveis. Neste artigo, propomos vários ensaios simples, rápido e acessível, estes ensaios têm sido validados por muitas publicações e são usados ​​rotineiramente para detectar ROS ou dos seus efeitos em células de mamíferos. Embora alguns desses ensaios detectar múltiplas ROS, outros detectam apenas um único ROS.

Watch this Scientific Journal Video about Production and Detection of Reactive Oxygen Species ROS in Cancers at JoVE.com

Production and Detection of Reactive Oxygen Species ROS in Cancers

Aqui nós propor métodos simples para testar e avaliar a presença de espécies reativas de oxigênio nas células.

Produção e Detecção de Espécies Reativas de Oxigênio (ROS) em Câncer

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

production-and-detection-of-reactive-oxygen-species-ros-in-cancers

Research

JoVE Journal

Medicine

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

69.8K visualizações.  Baylor College of Medicine. Aqui nós propor métodos simples para testar e avaliar a presença de espécies reativas de oxigênio nas células.

Vídeo: Production and Detection of Reactive Oxygen Species ROS in Cancers

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

Visualização e análise do fluxo sangüíneo e consumo de oxigênio na microcirculação hepática: aplicação para um modelo Hepatite Aguda

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

Medicina

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Luminescência de Cerenkov Imaging (CLI) para o Monitoramento Terapia de Câncer

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

Isolando e com cortes de bovinos artéria e veia mesentérica como Bioassay teste para vasoactividade no intestino delgado

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Avaliando Encefalopatia Espongiforme Transmissível Espécies Barreiras com uma<em&gt; In Vitro</em&gt; Prion Conversão Protein Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T &#945;S and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of &#945;SD (disease-associated &#945;S) in these mice, an ELISA was designed to specifically quantify &#945;SD in sick mice. Analysis of the central nervous system in this mouse model showed the presence of &#945;SD mainly in the caudal brain regions and the spinal cord. There were no differences in &#945;SD distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of &#945;SD immunoreactivity using an antibody against Ser129 phosphorylated &#945;S by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of &#945;S. The propagation of &#945;SD, suggesting the involvement of a &#8220;prion-like&#8221; mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

Detection of Disease-associated &amp;#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &amp;#945;-synuclein

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

Detecção de doença associada α-sinucleína por ELISA reforçada no cérebro de ratinhos transgénicos que sobre-expressam A53T Humano Mutante α-sinucleína

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

Disparo Reactive Gliose<em&gt; In Vivo</em&gt; Por um Stab Lesão Encéfalo anterior

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

In Vitro e In Vivo de deteção de Mitophagy em células humanas, C. Eleganse os ratos

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

Detecção baseada em plaquetas de óxido nítrico no sangue, medindo-se a fosforilação VASP

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

Mapeamento quantitativo de ventilação específica no pulmão humano usando ressonância magnética de prótons e oxigênio como agente de contraste

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When deoxygenated, HbS can polymerize, forming rigid rods of hemoglobin, resulting in the sickling of red blood cells (RBCs). These sickled RBCs have significantly reduced deformability, causing vaso-occlusion, which leads to numerous SCD-related clinical complications, including pain, stroke, and organ damage. RBC deformability is also reduced by RBC dehydration, resulting in dense red blood cells that are more likely to sickle. To date, there is not a single widely available, rapid, and reproducible laboratory assay capable of predicting the disease severity or directly monitoring the treatment effects for novel, non-fetal hemoglobin inducing therapies. In this study, we describe a protocol to measure RBC deformability as a function of pO2 that allows for the quantitation of sickling behavior in SCD patients. Oxygen gradient ektacytometry measures RBC deformability, expressed as the elongation index (EI), as a function of pO2. RBCs are exposed to a fixed shear stress of 30 Pa during one round of deoxygenation and reoxygenation. Six readout parameters are produced. Of these, the point of sickling (PoS), defined as the pO2 at which maximum EI (EImax) shows a 5% decrease, and minimum EI during deoxygenation (EImin) are the most informative, reflecting an individual patient&#8217;s pO2 at which sickling starts and the minimal deformability of a patient&#8217;s red blood cells, respectively. PoS is associated with an individual patient&#8217;s hemoglobin affinity for oxygen, whereas EImin shows a strong correlation with fetal hemoglobin levels. We conclude that oxygen gradient ektacytometry is a promising technique to monitor the treatment of patients with SCD, as a biomarker for anti-sickling agents in clinical and preclinical trials, and an important tool to study sickling behavior of RBCs from individuals with SCD and sickle cell traits.

Here, we present oxygen gradient ektacytometry, a rapid and reproducible method to measure red blood cell deformability in samples from patients with sickle cell disease under controlled deoxygenation and reoxygenation. This technique provides a way to study red blood cell sickling and to monitor sickle cell disease treatment efficacy.

Caracterização de Sickling durante desoxigenação automatizada controlada com o gradiente ektacytometry do oxigênio

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When ...