Este trabalho foi financiado pelo NIH National Heart, Lung, and Blood Institute concessão RO1 HL81248.

1. Cultura de Células endoteliais de aorta bovina (BAEC) <ol><li> Técnicas adequadas de assepsia foram seguidos. </li><li> Num meio de água do banho quente, sem antibióticos, a 37 ° C. </li></ol> Nota: O meio consiste em fenol meio Dulbecco modificado por Eagle livre (DMEM) com 4,5 g / L D-glucose, 4 mM de L-glutamina, 1% de não-aminoácidos essenciais, suplementado com 10% de soro fetal bovino (FBS) e 2,5 mg / L o factor de crescimento endotelial. <ol start="3"><li> Remover as células T75 balão contendo a partir da incubadora e limpar a superfície do balão com etanol 70% antes de colocá-lo dentro do capuz. </li><li> Remover o meio antigo usando um aspirador e lavar duas vezes com 5 ml de fosfato de Dulbecco tampão salina (DPBS). </li><li> Adicionar 2 ml de tripsina e esperar por 4-5 min para as células a separar enquanto periodicamente inspeccionar sob o microscópio. </li><li> Adicionar 3 ml do meio de misturar e repetidamente, utilizando uma pipeta de t separadoele células e criam uma suspensão mesmo. </li><li> Transferir 5 ml de meio com tripsina para um tubo de 15 ml e centrifugar a 121 g durante 5 min. </li><li> Remover o sobrenadante utilizando um aspirador. Adicionar 5 ml de DPBS e misture bem. Centrifugar a 121g durante 5 min. </li><li> Aspirar o sobrenadante e re-suspender o sedimento de células por adição de 6 ml de meio. </li><li> Em uma placa de 6 poços, adicionar 1 ml da suspensão de células a cada poço, em seguida, adicionar 1 ml de meio. Misturar a suspensão utilizando uma pipeta. </li><li> Rotular o placa e incubar durante a noite a 37 ° C e 5% de CO 2. </li></ol> 2A. Detecção de NO com Cell BAEC <ol><li> Técnicas adequadas de assepsia foram seguidos. </li><li> Remova a placa de 6 poços da incubadora e aspirar o meio a partir do primeiro poço. Lave as células duas vezes com DPBS 1 ml. </li><li> Adicionar 210 uL de 1,9 mM de sulfato de ferro hepta-hidratado (II) (FeSO 4 .7 H2O, preparar recentemente por dissolução de 0,8 mg em1 ml com DPBS CaCl2 e MgCl2) e uL 210 de amónio de N-metil-D-glucamina ditiocarbamato (DGM, preparar recentemente por dissolução de 2,7 mg em 500 ul com DPBS CaCl2 e MgCl 2) utilizando um rácio de 1:7 . (Nota:. Este rácio onde MGD excesso é usado tem sido convencionalmente empregue para a preparação de Fe 2 +-MGD complexa devido ao facto de que o rendimento do Fe 3 +-MGD é maximizada na presença de excesso de MGD A adição de ascorbato em solução para estabilizar o Fe 2 +-MGD não é necessário uma vez que a NO-3 Fe +-MGD formado é endogenamente reduzido para o EPR ​​detectável NO-Fe 2 +-MGD por ascorbato, hidroquinona, ou cisteína com uma eficiência de conversão de até a 99,9% .. O estado diamagnético baixo spin de Fe 2 + permite a detecção de NO utilizando células plana ou tubo capilar, sem a necessidade de um dispositivo de baixa temperatura) 15. </li><li> Swirl a suspensão resultante bem e acrescente 4,6 μl de ionóforo de cálcio (CAI) (preparado a partir de uma solução stock de 1,9 mM por dissolução de 1 mg em 1 ml de DMSO). </li><li> Solução Agitar o novo e incubar a 37 ° C durante 36 min, a fim de permitir ainda mais redução de NO-Fe 3 +-MGD para o EPR ​​detectável NO-Fe 2 +-MGD. </li><li> Recolher o sobrenadante (425 ul) em um tubo de Eppendorf e transferência para uma célula de EPR plana (ou para um tubo de 50 uL capilar). </li><li> Parâmetros de aquisição EPR ​​são: freqüência de microondas: 9,8 GHz; centro do campo: 3427 G; modulação de amplitude: 6,0 G; largura de varredura: 100 G; ganho do receptor: 1 x 10 5; potência de microondas: 10 mW; número total de verificações: 121; tempo de varredura: 10 s; tempo e constante: 20 ms (Nota: Uma vez que os parâmetros variam de um instrumento e condições experimentais para outro, portanto, apenas o campo de centro, a freqüência ea modulação de amplitude são os parâmetros mais importantes a considerar.). </li><li> Gravar os espectros à temperatura ambiente e os espectros de 2-D foram integclassificado para reduzir o ruído de fundo ea linha de base corrigido usando Bruker WinEPR software de processamento de dados ou software de processamento de dados. Para a quantificação de formação de aduto, parcelas padrão de concentração versus intensidade de sinal (ou área) pode ser construída usando um SNAP dador NO. </li></ol> 2B. eNOS Experiment Desacoplamento <ol><li> Remova a placa de 6 poços da incubadora e aspirar o meio a partir do segundo poço e lavar duas vezes com DPBS 1 ml. </li><li> Um doador de peroxinitrito, 5-amino-3-(4-morfolinil) -1,2,3-oxadiazolium cloreto de (SIN-1) foi utilizado para desacoplar eNOS 16. Adicionar 100 uL de 0,5 mm de SIN-1 (M r 206,6 g / mol, a partir de 10 mM de solução stock preparada por dissolução de 1 mg de SIN-1 em 500 ul de PBS sem iões Ca / Mg) e diluído para 2 ml com DPBS e 10% de FBS. </li><li> Incubar durante 2 horas a 37 ° C e 5% de CO 2. </li><li> Retirar a placa da incubadora e lavar duas vezes com DPBS. </li><li> Adicionar 210 uL de2,8 mM de FeSO 4 .7 H2O e 210 uL de 19,6 mM MGD recém-preparado de acordo com o procedimento acima mencionado. </li><li> Solução redemoinho e adicionar 4,6 ul de 1,9 mM CAI. </li><li> Solução Agitar o novo e incubar a 37 ° C durante 36 min. </li><li> Recolher o sobrenadante (425 ul) em um tubo de Eppendorf e transferência para uma célula de EPR plana. </li><li> Parâmetros de aquisição EPR ​​são: freqüência de microondas: 9,8 GHz; centro do campo: 3427 G; modulação de amplitude: 6 G; largura de varredura: 100 G; ganho do receptor: 1 x 10 5; potência de microondas: 10 mW; número total de verificações: 121; tempo de varrimento: 10 s; e constante de tempo: de 20 ms. </li><li> Gravar os espectros e os espectros de 2-D foram integradas para reduzir o ruído de fundo e de linha de base corrigido como mencionado acima. </li></ol> 3. Detecção de O 2 • - a partir de neutrófilos polimorfonucleares (PMNs) <ol><li> Os neutrófilos foram isolados a partir de amostra de sangue humano, como descrito anteriormente 17. </li><li> Fazer uma solução stock de 1 M DMPO * em PBS contendo 0,1 mM de dietilenotriamina-pentacético ácido (DTPA). DMPO tem um ponto de fusão de 25-29 ° C por isso é mais conveniente para pipetar DMPO líquido (densidade ~ 1,02 g / ml a 25 ° C) para um frasco de vidro (Nota: não utilizar frascos de plástico para a pesagem de desde DMPO puro reage com plástico). Congelado DMPO pode ser derretido, executando água morna para o frasco (Nota: não correr água quente como DMPO pode se decompor). </li><li> É importante a utilização de elevada pureza DMPO (&gt; 99%) desde algumas das armadilhas de spin comercialmente disponíveis contêm impurezas paramagnéticas, e portanto, é imperativo para executar espectro EPR de apenas a solução DMPO por si só (10 mM, neste caso). É crítico que nenhum sinal de fundo é evidente (ver Figura 3A). </li><li> Preparar solução de reserva (1 mg / ml) de forbol-12-miristato-13-acetato (PMA) em DMSO. Fazer alíquotas por diluição da solução a 10 ug / ml em PBS. </li><li> Em um 1.5 ml tubo Eppendorf, preparar uma solução com um volume total de 0,6 ml, seguindo a sequência da adição: ~ 10 6 células por ml de PMN, D-glucose (1 mg / ml) e albumina (1 mg / ml), 10 mM DMPO e 0,2 ug / ml de PMA. (Nota: PMA é o activador de radicais e deve ser adicionado por último). </li><li> Transferir a solução para uma célula de EPR plana. </li><li> Parâmetros de EPR condições de aquisição são: freqüência de microondas: 9,8 GHz; centro do campo: 3486 G; modulação de amplitude: 0,5 G; largura de varredura: 100 G; ganho do receptor: 5 x 10 5; número total de exames: 10; tempo de varredura: 30 s ; potência de microondas: 20 mW; e constante de tempo: ms 81. Para a quantificação de formação de aduto, parcelas padrão de concentração versus intensidade de sinal (ou área) pode ser construída usando os nitróxidos estáveis, tais como TEMPO ou 3-carboxílico-PROXYL. </li></ol> * Nota importante sobre a utilização de DMPO: Ao utilizar uma célula plana, pode-se aumentar a densidade de células, e assim aumentar o sinalintensidade do aducto de rotação, mas a meia-vida de O 2 • - aducto de DMPO é curto (t 1/2 ~ 1 min), que se decompõe para DMPO-OH. DMPO podem ser substituídos usando as mesmas concentrações de Empo, BMPO ou DEPMPO que estão disponíveis comercialmente para a estabilidade aumentada aducto com t 1/2 ~ 8 e 14 min, respectivamente. 4. Os resultados representativos Girar aprisionamento de NO radical foi realizada utilizando Fe 2 +-MGD 2A. Figura e 2B mostram nenhum sinal EPR de Fe 2 +-MGD ou mistura de Fe 2 +-MGD com a CAI, indicando que nenhum sinal de fundo NO origina a partir destes reagentes. BAEC após estimulação com a CAI, que liberta NO reage com o Fe 2 +-MGD para formar o aducto de rotação, NO-Fe 2 +-MGD, e mostra um sinal tripleto característica com valor hiperfina separação (hfsc) constante de uma N = 12,66 G e fator g de g = 2,040. (Figura 2C). O hfvalor sc foi determinada utilizando o programa de simulação WINSIM que pode ser baixado do site NIEHS Banco de Dados Software EPR. O hfsc experimental é consistente com o valor da literatura de um N = 12,70 G e G = 2,041 18 para o NO Fe-2 +-MGD aduto. Do mesmo modo, o efeito de SIN-1 em BAEC baixou a produção de NO, devido à eNOS desacoplamento como mostrado na Figura 2D. DMPO armadilha de spin foi usado para O 2 • - detecção. DMPO por si só não deu um sinal conforme mostrado nas Figuras 3A e confirmação de que a armadilha de rotação é livre de impurezas paramagnéticas. A Figura 3B é o espectro de DMPO e PMNs apenas, e da mesma forma, não há sinal detectável, sugerindo que o DMPO não causar activação do NADPH oxidase enzima. A Figura 3C mostra o sinal observado EPR ​​após estimulação de PMN por PMA. Os valores hfsc para este sinal foram determinados como sendo um N = 14,71 G, um ebeta-H = 11,40 G e uma γ-H = 1,25 G, e são consistentes com os valores na literatura de um N = 14,3 G, uma β-H = 11,7 G e uma γ-H = 1,3 G 19 para DMPO-ó 2 aducto H. <img src="/files/ftp_upload/2810/2810fig1.jpg" alt="A Figura 1" /> Figura 1. Fluxograma para a detecção de radicais a partir de neutrófilos e BAEC utilizando armadilhas de spin de EPR. (A) PMNs foram misturados com DMPO e PMA, ea mistura resultante transferida para uma célula de EPR plana para aquisição de dados. (B) BAEC foram cultivadas numa placa, e lavou-se com DPBS. A rotação armadilha Fe (MGD) 2 foi adicionado juntamente com CAI. A solução foi misturada cuidadosamente e incubados. A mistura foi transferida para uma célula de EPR plana para a EPR aquisição de dados. <img src="/files/ftp_upload/2810/2810fig2.jpg" alt="A Figura 2" /> Figura 2. Detecção EPR ​​de NO a partir de BAEC. (A) Espectro de Fe 2 +-MGD apenas (B) Espectro de Fe 2 +-MGD + CAI só. (C) Espectro de Triplet resultante a partir do NO aprisionamento por Fe 2 +-MGD usando CAI-estimulado células. (D) Espectro mostrando diminuição da produção de NO devido a 0,5 mM SIN-1 o tratamento de células. <img src="/files/ftp_upload/2810/2810fig3.jpg" alt="A Figura 3" /> Figura 3. Detecção EPR ​​de H 2 DMPO-O a partir de neutrófilos activados. (A) Espectro de 10 mM DMPO só. (B) Espectro de PMN sozinho na presença de 10 DMPO mM. (C) Espectro de PMN activado por PMA, na presença de 10 DMPO mM.

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EPR aprisionamento de rotação tem sido empregada em uma ampla variedade de aplicações biomédicas para quantificar e identificar os radicais livres. Aprisionamento de rotação é altamente sensível, capaz de detectar os radicais em concentrações que variam de nM a iM, tornando-o adequado para aplicação em sistemas biológicos. A formação do aduto paramagnético, NO-Fe 2 +-MGD, é a base de NO a detecção através de EPR. Fe 2 +-MGD reage com NO rapidamente 18 a uma ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Fenol meio DMEM livre Alta glicose 1X </td><td> GIBCO </td><td> 31053 </td><td></td></tr><tr><td> 0,25% de tripsina-EDTA </td><td> GIBCO </td><td> 25200 </td><td></td></tr><tr><td> L-Glutamina </td><td> Fisher Scientific </td><td> BP379-100 </td><td></td></tr><tr><td> MEM aminoácidos não essenciais </td><td> GIBCO </td><td> 11140 </td><td></td></tr><tr><td> Soro fetal de bovino </td><td> Atlanta Biologicals </td><td> S11550 </td><td></td></tr><tr><td> Factor de crescimento endotelial </td><td> Millipore </td><td> 02-102 </td><td></td></tr><tr><td> CAI </td><td> Enzo Ciências da Vida </td><td> A-23187 </td><td> Dissolve-se em DMSO </td></tr><tr><td> SIN-1 </td><td> Enzo Ciências da Vida </td><td> BML-CN245-0020 </td><td></td></tr><tr><td> DMPO </td><td> Dojindo Laboratories </td><td> D048-10 </td><td></td></tr><tr><td> FeSO 4 .7 H 2 O </td><td> Sigma Aldrich </td><td> 215.422-250G </td><td> Dissolve-se em PBS com Ca e Mg </td></tr><tr><td> MGD </td><td> Enzo Ciências da Vida </td><td> ALX-400-014-M050 </td><td> Dissolve-se em PBS com Ca 2 + e Mg 2 + </td></tr><tr><td> Células BAEC </td><td> Cell Systems </td><td> 2B2-C75 </td><td></td></tr><tr><td> DMSO </td><td> Fisher Scientific </td><td> BP231-100 </td><td></td></tr><tr><td> DPBS </td><td> Sigma Aldrich </td><td> D8537 </td><td></td></tr><tr><td> DPBS com CaCl2 e MgCl 2 </td><td> Sigma Aldrich </td><td> D8662 </td><td></td></tr><tr><td> Forbol miristato-acetato (PMA) </td><td> Sigma Aldrich </td><td> 79346-1mg </td><td></td></tr></tbody></table>

detection of nitric oxide and superoxide radical anion by electron paramagnetic resonance spectroscopy from cells using spin traps

Azoto reactivo / espécies de oxigénio (ROS / RNS) a baixas concentrações desempenhar um papel importante na regulação da função celular, de sinalização, e da resposta imune, mas em concentrações não regulados são prejudiciais para a viabilidade da célula 1, 2. Embora os sistemas vivos evoluíram com mecanismos de defesa antioxidantes endógenos e dietético para regular ROS geração, ROS são produzidos continuamente como natural, através da sub-produtos do metabolismo normal de oxigénio e pode causar danos oxidativos a biomoléculas, resultando em perda da função da proteína, a clivagem de ADN, ou lípido peroxidação 3, e, finalmente, ao estresse oxidativo levando a lesão ou morte celular 4. Anião radical superóxido (O2 • -) é o principal precursor de algumas das espécies mais altamente oxidantes conhecidos de existir em sistemas biológicos, tais como o peroxinitrito e radical hidroxilo. A geração de O 2 • - sinaliza o primeiro sinal de explosão oxidativa e, portanto, idetecção st e / ou a apreensão em sistemas biológicos é importante. Nesta demonstração, O 2 • - foi gerada a partir de neutrófilos (PMN). Através da estimulação com quimiotáctica de forbol-12-miristato-13-acetato (PMA), PMN gera O 2 • - através da activação de nicotinamida adenina dinucleótido fosfato oxidase (NADPH) 5. O óxido nítrico sintase (NO) que vem em três isoformas, como induzível-,-neuronal e endoteliais-SOE, ou iNOS, nNOS ou eNOS, respectivamente, catalisa a conversão de L-arginina a L-citrulina, utilizando NADPH para produzir NO 6 . Aqui, nós gerado NO a partir de células endoteliais. Sob condições de stress oxidativo, eNOS por exemplo, pode mudar de produzir NO para O 2 • - por um processo chamado de desacoplamento, o que se acredita ser causada pela oxidação do heme 7 ou o co-factor, tetrahidrobiopterina (BH 4) 8. Há apenas poucosmétodos fiáveis ​​para a detecção de radicais livres em sistemas biológicos, mas estão limitados por especificidade e sensibilidade. Aprisionamento de rotação é vulgarmente utilizado para a identificação de radicais livres e envolve a reacção de adição de um radical de uma armadilha de spin formando um aducto de spin persistente que pode ser detectado por espectroscopia de ressonância paramagnética electrónica (EPR). Os aductos vários radicais apresentam espectro característico que pode ser usado para identificar os radicais sendo geradas e pode fornecer uma grande quantidade de informação sobre a natureza e da cinética da produção de radical 9. As nitronas cíclicas, 5,5-dimetil-pirrolina-N-óxido, DMPO 10, o DEPMPO fosforilo-substituído 11, eo éster-substituído, Empo 12 e BMPO 13, têm sido largamente utilizados como armadilhas de spin - o spin último armadilhas exibindo mais uma meia-vida de O 2 • - aduto. Ferro (II)-N-metil-D-glucamina ditiocarbamato, Fe (DMG) 2 &lt;/&gt; Sub é vulgarmente usado para prender NO devido à elevada taxa de formação de aduto ea estabilidade elevada do adueto de rotação 14.

Watch this Scientific Journal Video about Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps at JoVE.com

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Electron paramagnético espectroscopia de ressonância (EPR) foi utilizado para detectar o óxido nítrico a partir de células endoteliais de aorta bovina e anião radical superóxido por neutrófilos humanos, utilizando ferro (II)-N-metil-D-glucamina ditiocarbamato, Fe (DMG)<sub&gt; 2</sub&gt; E 5,5-dimetil-1-pyroroline-N-óxido, DMPO, respectivamente.

Detecção de Óxido Nítrico e ânion radical superóxido por espectroscopia de ressonância paramagnética eletrônica a partir de células usando armadilhas para spins

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

detection-nitric-oxide-superoxide-radical-anion-electron-paramagnetic

Research

JoVE Journal

Biology

18.8K visualizações.  The Ohio State University. Electron paramagnético espectroscopia de ressonância (EPR) foi utilizado para detectar o óxido nítrico a partir de células endoteliais de aorta bovina e anião radical superóxido por neutrófilos humanos, utilizando ferro (II)-N-metil-D-glucamina ditiocarbamato, Fe (DMG) 2 E 5,5-dimetil-1-pyroroline-N-óxido, DMPO, respectivamente.

Vídeo: Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

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 ...

Biologia

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

Experimento Perfil bioenergética usando células C2C12 mioblastos

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level1. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography&#x2013;mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra2,3. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment4,5,6. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals7,8,9,10,11,12. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community13,14, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer2. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.

Concentração de metabólitos de comunidades de baixa densidade fitoplanctônicas para Metabolomics ambiental utilizando Espectroscopia de Ressonância Magnética Nuclear

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

Detecção de Vivo<em&gt; Escherichia coli</em&gt; O157: H7 Células por PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Dois fótons de imagem de cálcio intracelular<sup&gt; 2+</sup&gt; Manuseio e produção de óxido nítrico no endotélio e músculo liso células de uma aorta de rato isolada

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

Face Detection En de óxido nítrico e superóxido na camada endotelial das artérias intactas

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Desenvolvimento e Caracterização de<em&gt; In Vitro</em&gt; Rede de microvasos e medições quantitativas de endotelial [ca<sup&gt; 2+</sup&gt;]<sub&gt; i</sub&gt; E óxido nítrico Produção

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Detecção de formas modificadas da citosina Utilizando imunohistoquímica Sensível

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments &#8212; blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

Medindo nitrito e nitrato, metabolitos na Óxido Nítrico Pathway, em materiais biológicos utilizando o método de quimioluminescência

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&amp;#8226;) Probes, the geNOps, for Real-time Imaging of NO&amp;#8226; Signals in Single Cells

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

Aplicação de codificados geneticamente fluorescente óxido nítrico (NO •) Sondas, as geNOps, para imagens em tempo real de NO • Os sinais em células individuais

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...