Somos gratos ao Dr. Stephen Robinson e Ms Hania Czerwinska (Universidade Monash) para a generosa oferta de culturas de astrócitos.

1. Determinar ascorbato intracelular em células cultivadas <ol><li> Cultura de células e colheita <ol><li> Grow suspensão (por exemplo, eritroleucemia humana, K562) ou células aderentes (por exemplo, astrócitos primários) usando procedimentos padrão de cultura 19-21,32,38. Nota: para garantir células contêm ascorbato, carregar células cultivadas com ascorbato quer como ascorbato ou DHA 33,39. </li></ol></li><li> Criar um extrato celular contendo ascorbato <ol><li> Incubar de um número adequado de solução salina tamponada com fosfato de suspensão (PBS), lavou-se as células aderentes ou com 450 ul de células gelada permeabilização buffer [CEC; 0,1% (w / v) de saponina em PBS; 4 ° C]. Nota: determinar o número de células necessárias para se obter um valor de absorção dentro do intervalo linear do ensaio (ver abaixo) empiricamente pelo utilizador final. Nota: extratos de tecidos também pode ser usada (veja a discussão para mais detalhes). </li><li> Agitate células em gelo durante 10 minutos para garantir lise celular completa. </li><li> Criar um extracto intracelular contendo ascorbato clarificados removendo os restos celulares por centrifugação do ligado em bruto a 16000 × g durante 5 minutos numa microcentrífuga refrigerado (4 ° C). </li><li> Cuidadosamente remover 4 x alíquotas de 100 ul de cada amostra e adicionar a uma sequência horizontal de poços numa placa de 96 poços de fundo plano que contém ou 25 ul / poço de PBS (&quot;-AO&quot;) ou apenas 25 ul / poço de 45,5 L / ml de solução de L-ascorbato-oxidase (AO) em PBS (&quot;+ AO&quot;). Nota: este deve ser feita em paralelo com a preparação dos padrões (ver abaixo). </li></ol></li><li> Ascorbato padrão da curva de construção (deve ser construído de novo para cada ensaio) <ol><li> Prepara-se uma solução estoque de ácido ascórbico a 10 mM em PBS gelado. Nota: Verifique se a concentração de ácido ascórbico por espectrofotometria usando uma cuvete de quartzo com um 1cm de percurso a 265 nm (coeficiente de extinção = 14,5 mM -1 cm -1) 40. </li><li> Preparar-se cuidadosamente uma série de padrões de ascorbato entre 0 e 20 uM. </li><li> Em paralelo com a etapa 1.2.4, remover cuidadosamente 4 × alíquotas de 100 ul de cada padrão e adicionar a uma sequência horizontal de poços numa placa de 96 poços de fundo plano, que contém 25 ul / poço de PBS (&quot;-AO&quot;) ou um 45,5 solução estoque U / ml de AO em PBS (&quot;+ AO&quot;). Nota: 100 ul dos padrões de ascorbato irão conter 0-2 nmol de ascorbato. Por favor, note que o propósito do AO é para controlar a contribuição de redutores não-ascorbato de ferricianeto de redução. </li></ol></li><li> Determine ascorbato intracelular - Etapa 1: remover seletivamente ascorbato e quantitativamente oxidar ascorbato com ferricianeto <ol><li> Orbitalmente misturar a placa de 96 poços a 550 rpm à temperatura ambiente durante 5 min no escuro, cobrindo a placa em folha de alumínio. Nota: Tseu passo deve oxidar todos ascorbato no &quot;+ AO&quot; poços DHA. Os poços &quot;-ão&quot; não devem ser afetados. </li><li> Adicionar 50 mL de ferricianeto de potássio 3,5 mM (aqui referidos como &quot;ferricianeto&quot;) em PBS em todos os poços. O final [ferricianeto] = 1 mM. Nota: Utilize um multipipettor. </li><li> Orbitalmente misturar a placa a 550 rpm à temperatura ambiente durante mais 5 min no escuro. Nota: Esta etapa deve resultar na redução de ferricianeto de ferrocianeto de ascorbato numa razão estequiométrica de 2 moléculas de ferricianeto reduzidas por uma molécula de ácido ascórbico oxidado. </li><li> Imediatamente adicionar 25 ul de uma solução recentemente construído contendo 50% (v / v) de ácido acético e 30% (w / v) de ácido tricloroacético (TCA). Nota: Utilize um multipipettor. </li></ol></li><li> Determinar ascorbato intracelular - Passo 2: quantificar a quantidade de ferrocianeto formado 37 <ol><li> Adicione 100 ml de solução de ferrocianeto de 37 a determinação each bem. Nota: A solução de trabalho deve ser feita imediatamente antes da utilização: 2 ml de 3 M de Na-acetato (pH 6,0); 0,5 ml de ácido acético glacial (~ 17,4 M de ácido acético); 2 ml de ácido cítrico 0,2 M; 2 ml de 3,3 mM de FeCl3 em ácido acético 0,1 M; 1 ml de 30 mM Ferene-S. O volume final da solução de trabalho deve ser de 7,5 ml. </li><li> Orbitalmente misturar a placa no escuro a 550 rpm durante 30 min à temperatura ambiente. </li><li> Ler os valores de absorvância dos poços a 593 nm (isto é, o máximo de absorção do Fe (II) (Ferene-S) 3 complexo). </li><li> Calcule a quantidade de ácido ascórbico intracelular como nmoles ascorbato por milhão de células, inicialmente subtraindo os valores A 593 nm para poços a &#39;+ AO&#39; dos correspondentes - poços &#39;AO&#39; para cada amostra e, em seguida, interpolação na curva padrão ascorbato (veja o passo 1.3 ). Ao construir a curva padrão, traçar essa &quot;diferença de valor&quot; para cada padrão contra a quantidade de ascorbcomeu por poço. </li></ol></li></ol> 2. Determinação do ascorbato-efluxo de células cultivadas <ol><li> Cultura de células e colheita <ol><li> Crescer suspensão ou células aderentes como acima (veja o passo 1.1). Nota: realizar o ensaio a seguir em um formato de plataforma 24 poços com uma suspensão de células aderentes e para obter os melhores resultados. </li><li> Ressuspender as células em suspensão, as células aderentes ou de sobreposição, com 400 ul de solução salina tamponada com HEPES pré-aquecido, com ou sem cálcio e magnésio, e que contém 5 mM de D-glicose (HBS / D, pH 7,3, 37 ° C) nos poços de uma placa de 24 poços. Adicionar o mesmo volume de HBS / D para poços sem células especificadas em cada placa de 24 poços, para ser examinado. Nota: este último irá servir como controlos isentos de células para a reacção de referência (ver abaixo). </li></ol></li></ol> 3. Determinar a quantidade de ácido ascórbico Lançamentos <ol><li> As seguintes soluções de deve ser preparado com antecedência: 120U / ml AO em HBS / D (preparar fresco); 2,4 mM Ferene-S em HBS / D; 120 pM FeCl3 e 600 uM de Na-citrato em HBS / D (preparar imediatamente soluções de estoque mais concentrados). </li><li> Para iniciar a reação ferrireduction (volume final por poço deve ser de 600 mL), adicionar os seguintes volumes de reagentes aos poços individuais já contendo 400 mL de HBS / D: <ol><li> Adicionar 50 ul de AO (120 U / ml) ou HBS / D para emparelhado poços em triplicado. A concentração final AO deve ser de 10 U / ml. Nota: Etiqueta emparelhado poços como &quot;- AO&quot; e &quot;+ AO&quot;. </li><li> Misturar as placas com mistura suave orbital durante 5 min a 37 ° C. </li><li> Adicionar 50 ul de 2,4 mM Ferene-S para todos os poços. A concentração final Ferene-S deve ser de 200 uM. Misturar as placas como acima durante 5 min a 37 ° C. </li><li> Adicionar 50 ul de 120 uM recentemente preparada de citrato férrico. A concentração de citrato de ferro final e deve ser de 10 uM de ferro e citrato a 50 uM. Misture bem. </li></ol&gt; </li><li> Em três poços de controlo triplicadas, aspirar o meio sobrejacente e adicionar 600 mL HBS / D contendo 0,1% de saponina. Nota: Estes irão servir como &quot;100% de células-lise&quot; controlos de lactato desidrogenase (LDH), ensaio de libertação para ser conduzido em paralelo (ver abaixo). </li><li> Incubar durante 60 min no escuro a 37 ° C. </li><li> No final do ensaio de ascorbato-efluxo, aspirar-se rapidamente 500 mL de cada poço e adiciona ao apropriadamente marcada poços de uma placa de 24 poços. Nota: para as células em suspensão, inicialmente remover as células por centrifugação a 4 ° C. </li><li> Adicionar 300 mL alíquotas do sobrenadante para uma placa de 96 poços e, em seguida, ler a 593 nm. </li></ol> 4. Determinação de ascorbato Extracelular <ol><li> Leia os valores de absorbância dos poços a 593 nm. </li><li> Calcula-se a quantidade de ácido ascórbico extracelular como nmoles de ascorbato por mg de proteína (ou por milhão de células) tal como descrito para a determinação de ácido ascórbico intracelularno Passo 1.5.4. Nota: o utilizador final deve optimizar as condições de forma que a libertação de ascorbato não é limitada por ascorbato intracelular. </li></ol> 5. Determinação da LDH Lançamento <ol><li> Com os restantes 200 mL de solução extracelular a partir de cada amostra de realizar um ensaio de libertação de LDH 32,41 para determinar a extensão da lise celular. Nota: calcular como% da LDH releasable total. Determinar LDH libertável a partir das amostras que foram tratadas com 0,1% de saponina. </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+sensitive+assay+for+ascorbate+using+a+plate+reader.">A simple and sensitive assay for ascorbate using a plate reader.</a> Anal. Biochem. 365, 31-39 (2007).</li><li>Lane, D. J. R., Lawen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+sensitive+colorimetric+microplate+ferrocyanide+assay+applied+to+ascorbate-stimulated+transplasma+membrane+ferricyanide+reduction+and+mitochondrial+succinate+oxidation.">A highly sensitive colorimetric microplate ferrocyanide assay applied to ascorbate-stimulated transplasma membrane ferricyanide reduction and mitochondrial succinate oxidation.</a> Anal. Biochem. 373, 287-295 (2008).</li><li>Lane, D. J. R., Robinson, S. R., Czerwinska, H., Lawen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+Na+/H++exchangers+and+intracellular+pH+in+regulating+vitamin+C-driven+electron+transport+across+the+plasma+membrane.">A role for Na+/H+ exchangers and intracellular pH in regulating vitamin C-driven electron transport across the plasma membrane.</a> Biochem. J. 428, 191-200 (2010).</li><li>Corti, A., Casini, A. F., Pompella, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+pathways+for+transport+and+efflux+of+ascorbate+and+dehydroascorbate.">Cellular pathways for transport and efflux of ascorbate and dehydroascorbate.</a> Arch. Biochem. Biophys. 500, 107-115 (2010).</li><li>Laroff, G. P., Fessenden, R. W., Schuler, R. 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+electron+spin+resonance+spectra+of+radical+intermediates+in+the+oxidation+of+ascorbic+acid+and+related+substances.">The electron spin resonance spectra of radical intermediates in the oxidation of ascorbic acid and related substances.</a> J. Am. Chem. Soc. 94, 9062-9073 (1972).</li><li>Dringen, R., Kussmaul, L., Hamprecht, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detoxification+of+exogenous+hydrogen+peroxide+and+organic+hydroperoxides+by+cultured+astroglial+cells+assessed+by+microtiter+plate+assay.">Detoxification of exogenous hydrogen peroxide and organic hydroperoxides by cultured astroglial cells assessed by microtiter plate assay.</a> Brain Res. Brain Res. Protoc. 2, 223-228 (1998).</li><li>Lane, D. J. R., Lawen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplasma+membrane+electron+transport+comes+in+two+flavors.">Transplasma membrane electron transport comes in two flavors.</a> Biofactors. 34, 191-200 (2009).</li><li>Lin, S., Lin, D. C., Flanagan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+the+effects+of+cytochalasin+B+on+transport+and+motile+processes.">Specificity of the effects of cytochalasin B on transport and motile processes.</a> Proc. Natl. Acad. Sci. USA. 75, 329-333 (1978).</li><li>May, J. M., Qu, Z. C., Juliao, S., Cobb, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascorbic+acid+decreases+oxidant+stress+in+endothelial+cells+caused+by+the+nitroxide+tempol.">Ascorbic acid decreases oxidant stress in endothelial cells caused by the nitroxide tempol.</a> Free Radic. Res. 39, 195-202 (2005).</li><li>Avron, M., Shavit, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+and+simple+method+for+determination+of+ferrocyanide.">A sensitive and simple method for determination of ferrocyanide.</a> Anal. Biochem. 6, 549-554 (1963).</li></ol>

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Neste artigo, apresentamos dois ensaios de microplacas colorimétricos rápidos, específicos e relativamente sensíveis para a determinação de ácido ascórbico derivado dos compartimentos intra e extracelulares em células em cultura. Os ensaios podem ser concluída com acesso a equipamento de laboratório e reagentes. O reagente apenas moderadamente dispendioso necessário para o ensaio é de AO, que é essencial, uma vez que confere um elevado grau de especificidade para o analito L-ascorba...

A descoberta da natureza química do ácido ascórbico (vitamina C), e sua identificação como o &quot;fator anti-escorbuto&quot; há muito procurado, por Albert Szent-Györgyi e outros artigos publicados em 1928-1934 1 foram acontecimentos marcantes na história de bioquímica. De fato, essas descobertas contribuíram para Szent-Györgyi sendo agraciado com o Prêmio Nobel de Fisiologia ou Medicina em 1937. A suíte cada vez maior de papéis para ascorbato em fisiologia animal e vegetal, bem como a saúde humana, continuam a ser os temas de científico ativo investigação e controvérsia. L-ascorbato é um redutor fisiológico e abundante da enzima do cofactor em sistemas de mamíferos, e contribui para numerosas reacções enzimáticas bem definida envolvendo a hidroxilação do colagénio, carnitina e norepinefrina biossíntese, o metabolismo da tirosina e hormona peptídica amidação 2. Curiosamente, evide montagemdno sugere que o ácido ascórbico desempenha um papel na estimulação de outras dioxigenases dependentes de ferro, tais como os de prolilo e asparaginilo hidroxilases envolvidos na hidroxilação e direccionamento dos factores de hipoxia-inducible (HIFs) 1α e 2α 3. Um relatório recente sugere que o ácido ascórbico desempenha um papel na maturação de células T através da cromatina que afecta a sua actividade através de desmetilação para estimular as hidroxilases nucleares, proteínas de domínio (JmjC) Jumonji C; o último dos quais parecem exigir ascorbato para a atividade completa 4. Com efeito, a estimulação de tais enzimas por ascorbato parece ocorrer através de um mecanismo semelhante ao da estimulação por ascorbato de HIF e colagénio hidroxilases. Entre outros efeitos clássicos, ascorbato contribui significativamente para antioxidação celular como uma quebra de cadeia de captador de radicais solúveis em água e 5 para a reciclagem de membrana plasmática α-tocoferol (vitamina E), através da redução da α-tocopheroxyl radical 6, which é importante na proteção contra a peroxidação lipídica da membrana 7. É importante notar, embora a maioria dos mamíferos é capaz de síntese hepática de novo de ascorbato de D-glicose, primatas superiores, cobaias e alguns morcegos dependem de fontes dietéticas de vitamina 8. Isto é devido à inactivação do gene GULO, os ortólogos de que em mamíferos não afectadas codificam a enzima, γ-gulono-lactona oxidase 9-13. Esta enzima é necessária para a reacção final na biossíntese de ascorbato de glicose 13. Após a absorção mediada por transportador a partir do lúmen intestinal em seres humanos, o ácido ascórbico é distribuída por todo o corpo, o sistema circulatório. A vitamina é normalmente encontrado em sua forma reduzida em milimolares concentrações intracelular (com a notável exceção dos eritrócitos em que as concentrações são normalmente semelhantes aos da concentração plasmática vigente), e em conc micromolarentrations (por exemplo, 50-200 uM), na maioria dos fluidos extracelulares 14,15. Em condições fisiológicas, ascorbato normalmente sofre uma oxidação reversível de um elétron para o radical livre ascorbil (AFR, também conhecido como monodehydroascorbate ou semidehydroascorbate). Enquanto o AFR é relativamente estável radical 16, na ausência da sua rápida redução enzimática de um electrão de volta para o ácido ascórbico, dois AFRs pode promover dismutar para um ascorbato e um desidroascorbato (DHA) 9,13,17. Dentro do interior da célula, o produto de oxidação de dois electrões do ascorbato, DHA, pode ser rapidamente reduzida para trás pela glutationa-ascorbato e NAD (P) H-enzimática dependente e as reacções não-enzimáticas 13. Enquanto ele é classicamente aceito que apenas papel significativo do ascorbato no metabolismo do ferro é estimular a absorção dietética de ferro não-heme 18, nós e outros forneceram evidências strongly sugerindo que o ácido ascórbico desempenha um papel muito expandida no metabolismo deste metal. Em primeiro lugar, o ácido ascórbico que é liberado por células-ascorbato repleto parece desempenhar um papel importante na modulação da absorção de ferro não ligado à transferrina por células 19,20, e evidência muito recente indica que o ácido ascórbico também modula a absorção de ferro ligado à transferrina por células 21, o último dos quais corresponde a um grande fisiológico rota ferro-uptake 22. Ascorbato é essencial para a função normal do sistema nervoso central em mamíferos 23,24. Juntamente com o córtex supra-renal, glândula pituitária, do timo, da retina e do corpo lúteo, o cérebro contém altas concentrações de ascorbato em relação a outros tecidos do corpo 23,25-27. Além disso, a exposição de ambos os astrócitos 28,29 e células neuronais semelhante 30 ao glutamato é conhecido por provocar a libertação de ascorbato no espaço extracelular, onde a ascorbate é pensado para ajudar a proteger os neurônios contra a disfunção neuronal induzida pelo glutamato 31. Embora o mecanismo exato de glutamato induzida por liberação ascorbato de astrócitos é desconhecida, que recentemente forneceu evidências indicando o envolvimento de células inchaço causado pela captação de glutamato pela glutamato astrócitos e transportador de aspartato (GLAST, também conhecido transportador isoforma aminoácido excitatório 1 [EAAT1 ] em humanos) e conseqüente ativação de osmólito e ânions canais sensíveis ao volume (VSOACs) que são permeáveis ​​a pequenos ânions orgânicos, tais como ácido ascórbico 32. As identidades moleculares das condutas de membrana plasmática envolvidas na formação VSOAC continuam a ser identificadas 33,34. Embora muitos ensaios foram desenvolvidos para a determinação de ácido ascórbico em amostras biológicas, que incluem espectrofotométricos, fluorométricos e ensaios cromatográficos 35,36, existe uma grande variabilidade em especificidade, sensibilidade, interference por contaminantes químicos, faixa linear eficaz e estabilidade do analito endpoint. Além disso, outros factores importantes que influenciam a escolha do ensaio são a rapidez, a facilidade de utilização e acesso a equipamento relativamente especializadas tais como a cromatografia líquida de alto desempenho aparelho (HPLC). Aqui apresenta-se um ensaio de microplaca colorimétrico simples e altamente específico para a determinação de ácido ascórbico intracelular em células de cultura, assim como um ensaio separado para a determinação de ascorbato-efluxo a partir de células cultivadas. O último ensaio visa contornar o problema da subestimação da liberação ascorbato a partir de células devido ao rápido recaptação de ascorbato liberado pelos transportadores dependentes de ascorbato de sódio (SVCTs). Embora ambos os métodos têm aparecido em algumas de nossas publicações anteriores 19,20,32,37,38, este manuscrito fornece um conjunto explícito de instruções e orientações para a sua execução eficaz.

<table border="0" cellpadding="0" cellspacing="0" width="678">
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	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:284px;">Nunc 96-well flat-bottom plates</td>
			<td style="width:135px;">Thermo</td>
			<td style="width:127px;">269620</td>
			<td style="width:132px;">Any flat-bottom 96-well plate can be used</td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;width:284px;">Refrigerated benchtop microcentrifuge</td>
			<td style="width:135px;">Eppendorf&#160;</td>
			<td style="width:127px;">5415D</td>
			<td style="width:132px;">A non-refrigerated microcentrifuge that has been equilibrated to temperature in a cold room can also be used</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:74px;width:284px;">Refrigerated bench-top centrifuge</td>
			<td style="width:135px;">Eppendorf&#160;</td>
			<td style="width:127px;">5810R</td>
			<td style="width:132px;">Swing-bucket</td>
		</tr>
		<tr height="260">
			<td height="260" style="height:260px;width:284px;">Bio-Rad Benchmark Plus Microplate Spectrophotometer</td>
			<td style="width:135px;">Bio-Rad</td>
			<td style="width:127px;"></td>
			<td style="width:132px;">Any microplate spectrophotometer capable of reading at 593 nm can be used and is recommended. If a filter-based plate reader is used, choose the closest wavelength possible and use the standard-curve method.</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:284px;">Ependorf MixMate (microplate orbital mixer)</td>
			<td style="width:135px;">Eppendorf&#160;</td>
			<td style="width:127px;"></td>
			<td style="width:132px;">This is a very versatile and reliable microplate mixer and works very well for these assays</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">General-purpose buffers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Phosphate-buffered saline (PBS), pH 7.4</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:284px;">MOPS-buffered saline (MBS); 137 mM NaCl, 2.7 mM KCl, 15 mM MOPS-Na+, pH 7.3</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">MBS + 5 mM D-glucose (MBS/D)</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:284px;">HEPES-buffered saline + 5 mM D-glucose (HBS/D); 137 mM NaCl, 5.2 mM KCl, 1.8 mM CaCl2&#8226;2 H2O, 0.8 mM MgSO4&#8226;7 H2O, 5 mM D-glucose, 20 mM HEPES-Na+, pH 7.3)</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Cell permeabilisation buffer (CPB; 0.1% saponin in PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">General chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:284px;">L-ascorbic acid or sodium L-ascorbate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;"></td>
			<td style="width:132px;">Highest purity preparations should be obtained</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:284px;">Dehydro-L-ascorbic acid (DHA) dimer</td>
			<td>Sigma-Aldrich</td>
			<td>30790</td>
			<td style="width:132px;">Aqueous solutions theoretically yield 2 moles of DHA monomer per mole of DHA dimer</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:284px;">Cytochalasin B</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">C6762</td>
			<td style="width:132px;">Stock solutions prepared in DMSO or ethanol</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:284px;">Ascorbate oxidase (AO)</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">A0157</td>
			<td style="width:132px;">Stock solutions (120 U/ml) can be prepared in PBS or MBS and then frozen in aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Potassium ferricyanide (FIC)</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">455989</td>
			<td style="width:132px;">Trihydrate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Ferene-S (3-(2-Pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5&#8242;,5&#8242;&#8242;-disulfonic acid disodium salt)</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">92940</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Sodium L-glutamate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">L-glutamine</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Saponin</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">47036</td>
			<td style="width:132px;">Prepare a 0.1% stock solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;"></td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Stock solutions for intracellular ascorbate determination assay</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">3 M sodium acetate (pH 6.0)</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Glacial acetic acid</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">0.2 M citric acid</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:284px;">3.3 mM FeCl3 in 0.1 M acetic acid</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">30 mM ferene-S</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">50% (v/v) acetic acid + 30% (w/v) trichloroacetic acid (TCA)</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stock solutions for ascorbate-efflux assay</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">AO (120 U/ml)</td>
			<td></td>
			<td></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">2.4 mM ferene-S</td>
			<td></td>
			<td></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">0.12 mM FeCl3 in 0.6 mM sodium-citrate</td>
			<td></td>
			<td></td>
			<td style="width:132px;"></td>
		</tr>
	</tbody>
</table>

a rapid and specific microplate assay for the determination of intra- and extracellular ascorbate in cultured cells

A vitamina C (ácido ascórbico) desempenha várias funções importantes no metabolismo celular, muitos dos quais só vieram à luz nos últimos anos. Por exemplo, dentro do cérebro, o ácido ascórbico atua de forma neuroprotetor e neuromodulador que envolve ciclismo ascorbato entre astrócitos e neurônios vicinais - um relacionamento que parece ser crucial para ascorbato cérebro homeostase. Além disso, novas evidências sugerem fortemente que o ácido ascórbico tem um papel muito expandida na regulação do metabolismo do ferro celular e sistêmica do que é classicamente reconhecida. O crescente reconhecimento do papel fundamental do ascorbato na fisiologia celular e do organismo normal e desregulamentado exige uma gama de médio rendimento e de alta sensibilidade técnicas analíticas que podem ser executados sem a necessidade de equipamentos especializados altamente caro. Aqui nós fornecemos instruções explícitas para um meio-rendimento, ensaio de microplacas específico e relativamente barato para a determinação de boascorbato intra e extracelular th em cultura de células.

Determinação de ascorbato intracelular em células cultivadas de suspensão No primeiro ensaio (Figura 1), o ácido ascórbico intracelular é determinada, na sequência específicas de ascorbato (isto é, AO-sensível) redução de ferricianeto de ferrocianeto, utilizando a determinação altamente sensível do ferrocianeto de um processo publicado previamente 37. A detecção de ascorbato é baseado na quelação colorimétrico de ferro ferroso que é ge...

Watch this Scientific Journal Video about A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells at JoVE.com

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Ascorbato desempenha várias funções importantes no metabolismo celular, muitos dos quais só vieram à luz nos últimos anos. Descrevemos aqui um meio-débito, ensaio de microplacas específico e de baixo custo para a determinação de ambos ascorbato intra e extracelular em culturas de células.

Uma rápida e específica de microplaca de ensaio para a determinação de ascorbato intra-e extracelular em células cultivadas

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within ...

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Research

JoVE Journal

Biology

13.4K visualizações.  University of Sydney.  Ascorbato desempenha várias funções importantes no metabolismo celular, muitos dos quais só vieram à luz nos últimos anos. Descrevemos aqui um meio-débito, ensaio de microplacas específico e de baixo custo para a determinação de ambos ascorbato intra e extracelular em culturas de células. 

Vídeo: A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Contagem e determinação da viabilidade das células em cultura

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Biologia

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Clonogênica Ensaio: células aderentes

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Isolamento e Análise Funcional de mitocôndrias de células cultivadas e mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

Detecção de intracelular Expressão Gênica em células vivas de murino, humano e porcino Origem Usando nanopartículas marcados com fluorescência

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotoxina microinjeção no peixe-zebra ao modelo de lesão renal aguda

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

Isolamento de filtração de Ácidos Nucleicos: Um Método de extracção de DNA simples e rápido

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

A rápida, Método Scalable para o isolamento, estudo funcional, e Análise de matriz extracelular derivado de celular

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Uma interface de usuário gráfica para rastreamento assistido por software da concentração de proteínas em protrusões celulares dinâmicas

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

Expressão transitória e Celular Localização de proteínas recombinantes em células de cultura de insectos

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated &#945; subunit (HIF-1&#945;) and constitutively expressed &#946; subunit (HIF-1&#946;) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1&#945; stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1&#945; and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1&#945; and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions.

Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia.

Co-imunoprecipitação ensaio usando proteínas nucleares endógenas de células cultivadas em condições de hipóxicos

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...