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1. INS-1E células: manutenção e chapeamento
<ol><li>Manter células INS-1E numa incubadora de2 ˚C/5% CO 37 umidificado e culta com meio RPMI 1640 suplementado com 5% (v/v) inactivadas pelo calor fetal soro bovino, 2 mM L-glutamina, 10 mM HEPES, piruvato de sódio de 1 mM, 100 U/mL penicilina/estreptomicina solução de 50 µM de β-Mercaptoetanol. Cultura de células em meio RPMI 1640 completo de 10 mL (por placa), até atingirem a confluência de 80-90%, quando podem ser trypsinized e passadas ou usados para o ensaio de secreção de insulina.</li>
	<li>
Dia 1: Aspirar a mídia e lavar as células uma vez com 5 mL de PBS pré-aquecido. Adicione 0,5 mL de tripsina (0.025%) diluído 1:1 em 0,5 mL de PBS trypsinize as células e incubar durante 3-4 minutos a 37 ˚ c. Desactive a tripsina adicionando mídia completa 9 mL e a transferência de células para um tubo de centrífuga de 15 mL por pipetagem.</li>
	<li>Células de pelotas por centrifugação e ressuspender o centrifugado em aproximadamente 5 mL fresco meios de comunicação.</li>
	<li>Pegue 10 µ l de células re-suspensas e misture com 10 µ l corante vital de Trypan azul para verificar a viabilidade celular. Contar as células vivas e mortas em 10 µ l desta mistura usando um hemocytometer. Nível de viabilidade deve ser acima de 90%. Dilua as células re-suspensas em mídia fresca para 1 milhão de células por mL.</li>
	<li>Células de INS-1E de 0,5 mL de semente por bem em um poli-L-Lysine pré-revestido placa de 24, com uma densidade de 500.000 células/poço.</li>
	<li>
Dia 2: Remova a mídia 18-24h depois de chapeamento e adicionar 500 µ l/poço de mídia de RPMI 1640 fresco. Incube as células para outro 24h permitir que as células para se recuperar totalmente da etapa passaging prévia. Esse tempo adicional permite que as células a se espalhar na placa de cultura de tecidos.</li>
</ol>2. insulina secreção ensaio (dia 3)
<ol><li>Preparar o tampão KRB: 132,2 mM NaCl, 3,6 mM KCl, 5mm NaHCO3, 0.5 mM NaH2PO4, 0.5 mM MgCl2, 1,5 mM CaCl2e 0,001 g/mL albumina de soro bovino (BSA), pH 7,4.</li>
	<li>Aspire os meios de comunicação de células e lave duas vezes com PBS pré-aquecido.</li>
	<li>Para a etapa de fome de glicose, adicione 450 µ l/poço KRB (contendo BSA) sem glicose para 1h 37 ˚C/5% CO2.</li>
	<li>Durante a etapa de fome de glicose, preparar diluições em série da droga (s) em KRB contendo 200 mM glicose (10 x concentração).<ol>
<li>Prepare a droga em 10 x a concentração final em KRB suplementado com glicose 200 mM (também 10 x a concentração final de glicose). Se o estoque de drogas (e mais o adicionado 10 x glicose) é em DMSO, certifique-se de percentual de DMSO é mantido consistente durante todo o ensaio (idealmente final percentual inferior a 0,1% DMSO).</li>
		<li>Para tratamentos de dopamina e quinpirole, usar um intervalo de concentração de drogas de ensaio final de 100 µM a 100 pM (do maior para menor concentração), com o último ponto da resposta dose não contendo nenhuma droga. Para bromocriptina, use um intervalo de concentração de ensaio final de 10 µM para 22:00, com o último ponto da resposta dose sendo o controle de drogas.</li>
	</ol>
</li>
	<li>Depois da fome de glicose, adicione diluições em série drogas para o ensaio para produzir uma resposta de dose.</li>
	<li>Adicione 50 µ l/poço de cada diluição serial nos poços correspondentes (ensaio total volume 500 µ l).</li>
	<li>Para a etapa de estimulação de glicose, incubar as células com as diluições de série respectivas drogas (na presença de glicose de 20 mM) durante 90 minutos a 37 ˚C/5% CO2. Incluem um conjunto de poços de controle: (1) estimulação com glicose 20mm sozinho na ausência de qualquer droga adicional e (2) células que nem são estimuladas com drogas nem glicose (que fornece uma taxa basal de secreção).</li>
	<li>Após a etapa de estimulação, remova com cuidado os sobrenadantes (uso diretamente ou loja no 4 ˚ c). Nota: Um passo de centrifugação suave adicional 5 min (600 g de x, 1 min) pode ser introduzido neste momento para remover quaisquer células remanescentes nos sobrenadantes de ensaio.</li>
</ol>3. HTRF a secreção de insulina medida
<ol><li>Dilua o ensaio sobrenadantes 01:10 em KRB (sem BSA), de preferência em placas de 96 poços claros.</li>
	<li>Prepare a curva padrão de insulina para o ensaio de insulina HTRF (tabela 1).</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>Solução-padrão estoque 500 ng/ml</td>
			<td>Diluições em série</td>
			<td>Trabalhando [insulina] ng/ml</td>
		</tr>
<tr>
<td>STD 7</td>
			<td>30 µ l estoque + 140 µ l KRB</td>
			<td>150</td>
		</tr>
<tr>
<td>STD 6</td>
			<td>30 µ l STD 7 + 45 µ l KRB</td>
			<td>60</td>
		</tr>
<tr>
<td>STD 5</td>
			<td>30 µ l STD 6 + 45 µ l KRB</td>
			<td>24</td>
		</tr>
<tr>
<td>STD 4</td>
			<td>30 µ l STD 5 + 45 µ l KRB</td>
			<td>9.6</td>
		</tr>
<tr>
<td>DST 3</td>
			<td>30 µ l STD 4 + 45 µ l KRB</td>
			<td>3,84</td>
		</tr>
<tr>
<td>STD 2</td>
			<td>30 µ l STD 3 + 45 µ l KRB</td>
			<td>1,54</td>
		</tr>
<tr>
<td>STD 1</td>
			<td>30 µ l STD 2 + 45 µ l KRB</td>
			<td>0,61</td>
		</tr>
<tr>
<td>STD 0</td>
			<td>µ l 45 KRB</td>
			<td>0</td>
		</tr>
<tr>
<td colspan="3">Nota: Estoque STD é 500 ng/ml</td>
		</tr>
</tbody></table>Tabela 1. Diluições em série para fazer a curva padrão de insulina.
<ol start="3"><li>Adicione as amostras de curva padrão e os sobrenadantes de ensaio diluída para a placa HTRF. A medição de insulina secretada por HTRF pode efectuar-se em um poço-96 ou um formato de placa 384, mantendo em mente que o volume de ensaio deve ser re-ajustado. Uso 10 µ l/poço amostra em brancas 96 poços meia-área chapas ou 5 µ l/poço em uma placa de baixo volume, fundo redondo 384-poço branco (consulte Tabela de materiais).</li>
	<li>Preparar a mistura de anticorpo no buffer de deteção (ver Tabela de materiais) em um doador de 1:2 (cryptate) / relação do aceitador (XL-665). Nota: Mais detalhes específicos sobre o ensaio HTRF estão disponíveis do fabricante.</li>
	<li>Adicione a mistura de anticorpos para o ensaio em 30 µ l/poço (para um formato de 96-placa de ensaio) ou 15 µ l/poço (para um formato de ensaio de placa 384).</li>
	<li>Selar a placa e incubar à temperatura ambiente.</li>
	<li>Ler a placa depois de 2h, 4h, e/ou incubação durante a noite com anticorpos usando o leitor e o módulo de óptica de HTRF apropriado (337 665 620 nm) (ver Tabela de materiais e as instruções do fabricante). Definir o início da integração em 60 µs e o tempo de integração em 400 µs. uso 200 pisca por bem. Nota: Estes parâmetros foram baseados na utilização de nosso leitor específico. A leitura a 620 nm e 665 nm pode variar entre diferentes instrumentos. Esta é uma das razões que sugerimos usar a relação de 665/620. No cálculo deste rácio, quaisquer diferenças de potencial de leitor para leitor vão ser normalizadas em fornecem valores consistentes independentemente do instrumento utilizado para medir a HTRF.</li>
</ol>4. normalização e análise de dados
<ol><li>Calcular as concentrações de insulina dos poços de ensaio através de extrapolação de ratiometric leituras de fluorescência (665 nm/620 nm) para uma segunda ordem polinomial curva quadrática (Figura 2).</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57531/57531fig2.jpg"> Figura 2 : Curva padrão insulina. Estoque de insulina humana de concentrações conhecidas foi usado para gerar a curva padrão de insulina. Os rácios HTRF resultantes (665 nm / 620 nm) foram plotados contra as concentrações de insulina. Os dados foram o melhor ajuste para uma segunda curva polinomial quadrático de ordem (R2 = 0.99996). Esta é uma curva padrão representativa. Barras de erro = SEM. <a href="/files/ftp_upload/57531/57531fig2large.jpg" target="_blank">, por favor clique aqui para ver uma versão maior desta figura.</a>
<ol start="2"><li>Com os dados extrapolados como ng/mL de insulina secretada, normalize (insulina secretada em resposta ao aumento da concentração de ligante) para o valor médio de poços de teste a % máxima de insulina secreção (condição sozinho de glicose 20 mM).</li>
	<li>Use o ajuste de curva (R2) de uma única experiência para calcular a variação de intraplate. Dentro do experimento, derivado dos valores individuais de2 R as duplicatas intra experimentais, permitindo o cálculo do erro padrão da média para a curva de ajuste.</li>
	<li>Para determinar a variação interplate, use os dados de pelo menos três experimentos individuais para calcular o erro padrão da média para o valor de2 R da curva coletiva.</li>
</ol>

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Agradecemos Nicolas Pierre (Cisbio bioensaios) para conselhos úteis e Dr. Pierre Maechler (Universidade de Genebra) generosamente, fornecendo células INS-1E. Este trabalho foi apoiado pelo financiamento do departamento de defesa (grant PR141292 a Z.F.) e John F. e Nancy A. Emmerling fundo da Fundação de Pittsburgh (a Z.F.).

O ensaio de insulina HTRF descrito aqui oferece um sistema rápido e eficiente para medir a secreção de insulina de um sistema de baseados em células cultivada. Entre suas vantagens mais importantes, neste ensaio oferece um sinal baixo fundo devido a alta relação sinal-ruído. Além disso, confirmamos que o sinal HTRF é estável durante longos períodos de tempo (> 24 h)7. No entanto, desde que os anticorpos monoclonais de ligação de insulina rapidamente chegar a saturação de ligação a...

A regulação do metabolismo energético é aperfeiçoá-lo por um importante hormônio anabólico, a insulina. Insulina é sintetizada e liberada por células-beta do pâncreas em resposta aos níveis de glicose extracelular aumentado. A insulina liberada provoca a absorção de glicose pelos tecidos sensíveis à insulina1,2. Fisiologicamente, isto está ligado à elevação da concentração de glicose após uma refeição, seguida de secreção de insulina para regular a absorção de glicose. Perturbações na homeostase de glicose levam a deficiências metabólicas, culminando na resistência à insulina e, finalmente, no início da43,2,do diabetes tipo 2.
Embora a secreção de insulina tem sido extensivamente estudada, seus mecanismos regulatórios permanecem mal compreendidos. Uma área crítica de investigação tem sido a identificação de novos moduladores de secreção de insulina em células beta5,6,7,8. Estes estudos requerem uma melhor compreensão da relação de acoplamento entre a estimulação de glicose e a secreção de insulina. Portanto, a capacidade de precisão de monitorar e quantificar os níveis de secreção de insulina glicose-estimulada (GSIS) tem sido essencial. Até à data, no entanto, apenas um número limitado de métodos estava disponível para permitir a quantificação de GSIS usando linhas de células secretoras de insulina e/ou de ilhotas pancreáticas. Um é o radioimunoensaio (RIA), que utiliza anticorpos e insulina radioisótopo-tag. As principais limitações desta abordagem incluem questões de segurança devido à manipulação e eliminação de materiais radioactivos. Além disso, este método é trabalhoso, envolvendo múltiplos lavagem longa e etapas de incubação. Ensaio imunoenzimático (ELISA) é outra abordagem dispendiosa e trabalhosa que utiliza anticorpos para a deteção de insulina. Variação em afinidades de anticorpo e da eficiência de reconhecer a insulina estão limitando fatores desse método e pode afetar a reprodutibilidade dos resultados. ELISA, nem RIA foi projetado para experimentos de alta produtividade. AlphaScreen é um ensaio homogêneo usado para detectar e medir os níveis de secreção de insulina. AlphaScreen tecnologia baseia-se na conversão de oxigênio ambiente em um estado singlete oxigênio excitado que pode reagir com espécies quimioluminescente, resultando na geração de quimioluminescência. Porque o ensaio é homogêneo, muitas das etapas lavagem associadas a RIA e ELISA são eliminadas. No entanto, a instabilidade do sinal devido à natureza da reação é um fator limitante que possam afectar a leitura do ensaio. (TR-pinça, desenvolvido pela Heyduk e colegas9, é outra abordagem homogénea para medição de insulina com base na ligação de dois anticorpos separados para diferentes epitopos na molécula de insulina. Os anticorpos são cada DNA encalhado quimicamente ligado à dupla com curto complementares único encalhado DNA saliências. Ligação dos anticorpos à insulina reúne-os e leva a um duplex de DNA encalhado dobro. Cada anticorpo também está associado com um fluoróforo dador ou aceitador respectivo, e a associação do duplex de DNA reúne estes fluorophores para gerar a transferência de energia de ressonância de Förster (FRET). Uma limitação potencial de TR-pinça, baseia-se no entanto, com o traste em si. A incapacidade de dissipar rapidamente fluorescência de fundo durante a reação de FRET pode levar a níveis relativamente elevados de fluorescência de fundo e um baixo sinal à relação de ruído dentro do ensaio. Portanto, ainda existe uma necessidade para um ensaio confiável, robusto e econômico para quantificar GSIS em uma maneira de alta produtividade.
Avanços recentes em Biofísica culminaram no desenvolvimento de um ensaio de transferência (HTRF) com base de ressonância de energia homogênea fluorescência tempo-resolvido. Especificamente, enquanto a energia de transferência dentro o ensaio pode ser descrito como baseado no traste, mais precisamente, HTRF depende da luminescência energia ressonância transferência (LRET)10 que é non-radiative transferência de energia entre o doador e o aceptor espécie de11,12,13. Esta distinção é importante, desde o momento de uma fluorescência ou têmpera interação com base de FRET é muito diferente do que é para LRET, embora os mesmos tipos de retenção podem ser usados para FRET e LRET. Além disso, o uso de terras raras lantanídeos cryptate compostos tais como o európio ou térbio cryptate em HTRF produz fluorescência longa meia-vida de12,14. Isto oferece a vantagem exclusiva da introdução de um atraso de tempo (microssegundos por) entre a excitação do doador e a medição das emissões do aceitador (isto é, tempo-resolvido do ensaio). Este atraso do tempo permite tempo suficiente para que a fluorescência de fundo a dissipar-se antes da medição de fluorescência de emissão aceitador. Consequentemente, a leitura é livre de fluorescência não-específica e assim, uma alta relação sinal-ruído é alcançada (Figura 1). Além disso, a natureza homogênea de HTRF elimina a necessidade de etapas de lavagem para lavar a espécie desacoplada, fazendo o ensaio muito mais rápido do que os métodos baseados em RIA ou ELISA.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57531/57531fig1.jpg"> Figura 1: esquemático do mecanismo para a deteção de insulina HTRF. Dois anticorpos monoclonais independentemente gerados especificamente reconhece e vincule a insulina em locais separados. Estes anticorpos são conjugados para o doador de cryptate de európio ou o aceitador de XL665. Excitação do doador em 337 resultados de nm em emissão a 620 nm. A transferência de energia resultante faz com que XL665 emitir um comprimento de onda mais longos, 665 nm. <a href="/files/ftp_upload/57531/57531fig1large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
Aqui, nós fornecemos um protocolo detalhado para usar uma abordagem baseada em HTRF para determinar os níveis de GSIS de células INS-1E, uma bem estabelecida insulina secretoras derivado de células beta rato insulinoma célula linha15. Além disso, este ensaio pode ser utilizado para identificar o perfil farmacológico de reguladores moleculares da secreção de insulina. Aplicamos este ensaio de insulina com base em HTRF para examinar a dopamina D2-como o Regulamento do receptor de GSIS. Cada vez mais estudos têm revelado que o neurotransmissor dopamina é um importante regulador do GSIS8,16,17,18,19,20, 21 , 22. dopamina afeta GSIS de maneira autócrina/parácrina negativa através de ações sobre a dopamina D2-como os receptores (D2, D3, receptores de4 D) expressados na superfície de células pancreáticas beta8 , 16 , 19. usando este ensaio, confirmar o papel da dopamina como um regulador negativo de GSIS e demonstrar que a dopamina D2-como receptor agonistas bromocriptina e quinpirole também reduzem GSIS.

<table border="0" cellpadding="0" cellspacing="0" width="773">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-well white half area plate</td>
			<td style="width:104px;">Greiner Bio-One</td>
			<td style="width:139px;">82050-042</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">384-well white low-volume, round-bottom plate</td>
			<td style="width:104px;">Corning</td>
			<td>15100157</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Insulin High Range Kit</td>
			<td style="width:104px;">Cisbio Bioassays</td>
			<td style="width:139px;">62IN1PEH&#160;</td>
			<td>10,000 test HTRF-based insulin assay kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PHERAstar FSX plate reader</td>
			<td style="width:104px;">BMG Labtech</td>
			<td>FSX</td>
			<td>Plate reader adapted for HTRF-based assay readings</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Plate sealers</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:139px;">DY992</td>
			<td>To seal plate while antibodies incubate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hemocytometer</td>
			<td style="width:104px;">Hausser Scientific</td>
			<td style="width:139px;">3100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI Medium 1640 (1x)</td>
			<td style="width:104px;">Gibco&#160;</td>
			<td style="width:139px;">11875-093</td>
			<td>[+] L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin 0.05%&#160;</td>
			<td style="width:104px;">Corning</td>
			<td style="width:139px;">25-052-CI</td>
			<td>0.53 mM EDTA, (-) sodium bicarbonate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DPBS (Dulbecco&#39;s phosphate buffered saline)</td>
			<td style="width:104px;">Corning</td>
			<td style="width:139px;">21-031-CV</td>
			<td>Without calcium and magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:104px;">Corning</td>
			<td style="width:139px;">35-010-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:104px;">Gibco&#160;</td>
			<td style="width:139px;">156-30-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium pyruvate</td>
			<td style="width:104px;">Gibco&#160;</td>
			<td style="width:139px;">11360070</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicillin/Streptomycin solution 100x</td>
			<td style="width:104px;">Corning</td>
			<td style="width:139px;">30-002 CT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-mercaptoethanol</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">M1348</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan Blue Stain (0.4%)</td>
			<td style="width:104px;">Gibco&#160;</td>
			<td style="width:139px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dopamine hydrochloride</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">8502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bromocriptine mesylate</td>
			<td style="width:104px;">Tocris</td>
			<td style="width:139px;">427</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">(-)-Quinpirole hydrochloride</td>
			<td style="width:104px;">Tocris</td>
			<td style="width:139px;">1061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:104px;">Calbiochem</td>
			<td style="width:139px;">12659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poly-L-Lysine</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">P4832</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glucose</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">G7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO (Dimethyl sulfoxide)</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">276855</td>
			<td></td>
		</tr>
	</tbody>
</table>

homogeneous time-resolved f&#246;rster resonance energy transfer-based assay for detection of insulin secretion

A detecção de secreção de insulina é fundamental para elucidar os mecanismos de secreção regulamentado, bem como em estudos de metabolismo. Apesar de inúmeros ensaios de insulina já existem há décadas, o recente advento da tecnologia de Förster ressonância energia de transferência (HTRF) de tempo-resolvido homogênea simplificou significativamente estas medições. Isto é um ensaio óptico rápido, econômico, reprodutível e robusto depende dos anticorpos conjugados a brilhante fluorophores com emissão de longa duração, que facilita o tempo-resolvido Förster ressonância transferir energia. Além disso, a detecção de insulina HTRF é favorável para o desenvolvimento de ensaios de rastreio com throughput alto. Aqui usamos o HTRF para detectar a secreção de insulina em células INS-1E, uma linha de celular insulinoma-derivado de rato. Isso nos permite estimar os níveis basais de insulina e suas mudanças em resposta à estimulação de glicose. Além disso, nós usamos este sistema de deteção de insulina para confirmar o papel da dopamina como um regulador negativo da secreção de insulina glicose-estimulada (GSIS). De forma semelhante, outros de dopamina D2-como agonistas dos receptores, quinpirole e bromocriptina, reduzem GSIS de forma concentração-dependente. Nossos resultados destacam o utilitário do formato HTRF insulina ensaio para determinar o papel de numerosas drogas em GSIS e seus perfis farmacológicos.

Nós validado o nosso ensaio HTRF de insulina, gerando uma curva padrão de insulina, usando insulina humana purificada padrões predefinidos concentrações (Figura 2). Geração da curva padrão nos permitiu extrapolar as leituras de fluorescência ratiometric e, portanto, para determinar os níveis de insulina secretada em resposta aos tratamentos de drogas (Figura 2). Intraplate variação para o encaixe de curva foi mínima ...

Watch this Scientific Journal Video about Homogeneous Time-resolved FÃƒÂ¶rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion at JoVE.com

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Aqui, apresentamos homogênea tempo resolvido FRET (HTRF) como um método eficiente para detecção rápida de insulina secretada de células.

Förster homogênea de tempo-resolvido ressonância energia baseada em transferência ensaio para detecção de secreção de insulina

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

homogeneous-time-resolved-frster-resonance-energy-transfer-based

Research

JoVE Journal

Medicine

9.1K visualizações.  University of Pittsburgh. Aqui, apresentamos homogênea tempo resolvido FRET (HTRF) como um método eficiente para detecção rápida de insulina secretada de células.

Vídeo: Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

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

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

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

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

Medicina

Improving IV Insulin Administration in a Community Hospital

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose concentrations, even in non-diabetic patients, predicts poor outcomes.1-4 The 2008 consensus statement by the American Association of Clinical Endocrinologists (AACE) and the American Diabetes Association (ADA) states that "hyperglycemia in hospitalized patients, irrespective of its cause, is unequivocally associated with adverse outcomes."5 It is important to recognize that hyperglycemia occurs in patients with known or undiagnosed diabetes as well as during acute illness in those with previously normal glucose tolerance.
The Normoglycemia in Intensive Care Evaluation-Survival Using Glucose Algorithm Regulation (NICE-SUGAR) study involved over six thousand adult intensive care unit (ICU) patients who were randomized to intensive glucose control or conventional glucose control.6 Surprisingly, this trial found that intensive glucose control increased the risk of mortality by 14% (odds ratio, 1.14; p=0.02). In addition, there was an increased prevalence of severe hypoglycemia in the intensive control group compared with the conventional control group (6.8% vs. 0.5%, respectively; p&lt;0.001). From this pivotal trial and two others,7,8 Wyoming Medical Center (WMC) realized the importance of controlling hyperglycemia in the hospitalized patient while avoiding the negative impact of resultant hypoglycemia. 
Despite multiple revisions of an IV insulin paper protocol, analysis of data from usage of the paper protocol at WMC shows that in terms of achieving normoglycemia while minimizing hypoglycemia, results were suboptimal. Therefore, through a systematical implementation plan, monitoring of patient blood glucose levels was switched from using a paper IV insulin protocol to a computerized glucose management system. By comparing blood glucose levels using the paper protocol to that of the computerized system, it was determined, that overall, the computerized glucose management system resulted in more rapid and tighter glucose control than the traditional paper protocol. Specifically, a substantial increase in the time spent within the target blood glucose concentration range, as well as a decrease in the prevalence of severe hypoglycemia (BG &lt; 40 mg/dL), clinical hypoglycemia (BG &lt; 70 mg/dL), and hyperglycemia (BG &gt; 180 mg/dL), was witnessed in the first five months after implementation of the computerized glucose management system. The computerized system achieved target concentrations in greater than 75% of all readings while minimizing the risk of hypoglycemia. The prevalence of hypoglycemia (BG &lt; 70 mg/dL) with the use of the computer glucose management system was well under 1%.

When compared to the previous paper protocol, implementation of a computerized glucose management system results in a substantial increase in blood glucose concentration measurements within the target range. Using a computerized glucose management system to monitor blood glucose levels, decreases in severe hypoglycemia (&lt;40 mg/dL), clinical hypoglycemia (&lt;70 mg/dL), and hyperglycemia (&gt;180 mg/dL) also can be observed.

Melhorar IV A administração de insulina em um hospital comunitário

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose ...

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

Intraoperatória Detecção de Endometriose Sutil: um novo paradigma para a detecção e tratamento de dor pélvica associada com a perda de integridade Peritoneal

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

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

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

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

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

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

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function.
Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions.
Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.

Transcellular protein interactions are important determinants of pancreatic beta-cell function. Detailed here is a method&#x2014;adapted from a coculture model of synaptogenesis&#x2014;for investigating how specific transmembrane proteins influence insulin secretion. Transfected HEK293 cells express proteins of interest; beta cells do not need to be transfected or otherwise directly perturbed.

Análise de co-cultura da Relação de proteínas extracelulares que afetam secreção de insulina por células beta pancreáticas se

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

Combinado de microscopia Intravital e ultra-sonografia contraste aprimorado do membro posterior Mouse para estudar a vasodilatação induzida por insulina e perfusão muscular

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

Cardiac Magnetic Resonance Imaging at 7 Tesla

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher magnetic field strengths and potentially provides improved signal contrast and spatial resolution. While promising results have been achieved, ultra-high field CMR is challenging due to energy deposition constraints and physical phenomena such as transmission field non-uniformities and magnetic field inhomogeneities. In addition, the magneto-hydrodynamic effect renders the synchronization of the data acquisition with the cardiac motion difficult. The challenges are currently addressed by explorations into novel magnetic resonance technology. If all impediments can be overcome, ultra-high field CMR may generate new opportunities for functional CMR, myocardial tissue characterization, microstructure imaging or metabolic imaging. Recognizing this potential, we show that multi-channel radio frequency (RF) coil technology tailored for CMR at 7 Tesla together with higher order B0 shimming and a backup signal for cardiac triggering facilitates high fidelity functional CMR. With the proposed setup, cardiac chamber quantification can be accomplished in examination times similar to those achieved at lower field strengths. To share this experience and to support the dissemination of this expertise, this work describes our setup and protocol tailored for functional CMR at 7 Tesla.

The sensitivity gain inherent to ultrahigh field magnetic resonance holds promise for high spatial resolution imaging of the heart. Here, we describe a protocol customized for functional cardiovascular magnetic resonance (CMR) at 7 Tesla using an advanced multi-channel radio-frequency coil, magnetic field shimming and a triggering concept.

Ressonância magnética cardíaca na Tesla 7

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

Estudando o sinal de insulina hipotalâmico a intolerância de glicose periférica com um sistema de infusão contínua de drogas dentro do cérebro de rato

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis and septic shock. The early identification of an infectious process that is rapidly evolving to a critical illness might prompt a quicker and more intensive treatment, thereby potentially leading to better patient outcomes. The Endotoxin Activity (EA) assay can be used at the bedside as a reliable biomarker of systemic endotoxemia. The detection of elevated endotoxin activity levels has been repeatedly shown to be associated with an increased disease severity in patients with sepsis and septic shock. The assay is quick and easy to perform. Briefly, after sampling, an aliquot of whole blood is mixed with an anti-endotoxin antibody and with added LPS. Endotoxin activity is measured as the relative oxidative burst of primed neutrophils as detected by chemioluminescence. The assay&#39;s output is expressed on a scale from 0 (absent) to 1 (maximal) and categorized as &#8220;low&#8221; (&#60;0.4 units), &#8220;intermediate&#8221; (0.4&#8211;0.59 units), or &#8220;high&#8221; (&#8805;0.6 units). The detailed methodology and rationale for the implementation of the EA assay are reported in this manuscript.

We hereby present a protocol to measure at the bedside the endotoxin activity of human whole blood samples. The Endotoxin Activity assay is a simple test to perform and may be a useful biomarker in critically ill patients with sepsis.

Ensaio da atividade da endotoxina para a deteção do endotoxemia do sangue inteiro em pacientes criticamente doentes

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

Os mecanismos subjacentes a secreção do hormônio Gut usando o isolado perfundido intestino de rato

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...