Os autores gostariam de agradecer o Sr. Richard Zink e Todd Wiernicki para treinamento e orientação metodológica ensaio. Agradecemos também a John Paul Spence para a leitura cuidadosa e sugestões editoriais. Este trabalho foi financiado pelo Instituto Nacional de Saúde MH 060397, Brain and Behavior Research Foundation, e Eli Lilly and Company por meio do Programa de Pesquisa Prêmio Lilly (LRAP).

1. Expansão e criopreservação de células Ensaio Pronto <ol><li> Cultura de CHO-K1-DRD 2L células (CHO-D 2L) sobre uma cultura de células prato 15 centímetros 2 em meio F12 de Ham enriquecido com 1,0 uM de L-glutamina, 800 ug / mL de G418, 300 ug / higromicina ul, 100 U / ul penicilina, 100 ug / mL de estreptomicina, e 10% de soro fetal bovino (FBS). </li><li> Incubar as células a 37 ° C numa incubadora humidificada com 5% de CO2 até as células são 90-95% confluentes. Lavar as células com 10 uL de Tampão Fosfato Salino (PBS), e colher as células por adição de 3 ul de tampão de dissociação de células durante 5 min a 37 ° C. Ressuspender as células utilizando 12 mL dos meios de cultura e contagem de células usando exclusão do azul de tripan. </li><li> Centrifugar a suspensão de células a 500 xg durante 5 min à temperatura ambiente. Aspirar o sobrenadante e ressuspender o sedimento de células em 5 mL de meio de congelação (10% de dimetil sulfóxido, 90% FBS). Dilui-se a suspensão de células para se conseguir a concentração desejada de células (por exemplo, 1-20 x 10 6 células / mL). </li><li> Alíquota 1,0 mL da solução de célula para cada cryovial. Incubar as criotubos em um recipiente de congelação de células a -80 ° C durante a noite. Transferir os criotubos para um líquido N2 tanque para o armazenamento de longo prazo. </li></ol> 2. Cultivo de células Pronto Ensaio (e transfecção reversa opção) <ol><li> Rapidamente descongelar um criotubo gelado de células em um banho de água a 37 C º. Uma vez que as células são descongelados, transferir as células para um tubo cônico de 15 mL contendo 9 mL de Opti-MEM, e misturar invertendo o tubo 3-5x. </li><li> Centrifugar as células a 500 xg durante 5 min à temperatura ambiente. Aspirar o sobrenadante e ressuspender as células em 1 ml de Opti-MEM. </li><li> Contar as células usando exclusão com azul de tripano para determinar a viabilidade das células e diluir as células viáveis, conforme necessário (por exemplo. De 3 x 10 5 células / mL de concentração) emOpti-MEM. Placa 10 uL / ​​poço de células em cultura de tecidos tratados placa de 384 poços com uma pipeta multicanal. </li><li> Fazer diluições seriadas de AMPc em Opti-MEM para gerar uma curva padrão para estimar a produção de cAMP por as células (de acordo com a recomendações do fabricante - ver Secção 7). Adiciona-se 10 uL / poço dos padrões de cAMP à placa Nota:. Uma curva padrão único pode ser preparado sobre uma placa separada ou poços em branco sobre uma das placas de ensaio. </li><li> Centrifugar a placa a 100 g durante 15 segundos a temperatura ambiente, e incubar a 37 ° C numa incubadora humidificada com 5% de CO 2 durante 1 hora. </li></ol> 3. SiRNA transfecção opção Inverter * Esta secção é opcional e relevante para a Figura 3. <ol><li> Prepara-se uma solução de siRNA em DDH RNase 2 O (por exemplo, de 0,4 pmol / ul). Adicionam-se 5 ul a poços individuais de placas de 384 poços, e centrifugar a placa a 100 xg por 15 sic à temperatura ambiente. </li><li> Diluir Lipofectamina 2000, em Opti-MEM por um fator de 0,006 (por exemplo, adicionar 6 mL de Lipofectamina 2000 a 1000 l de Opti-MEM), e misture por pipetagem cima e para baixo. </li><li> Incubar a solução Lipofectamine 2000/Opti-MEM durante 5 min à temperatura ambiente. Adicionar 5 ml da solução de Lipofectamine 2000/Opti-MEM diluída para poços individuais de placas de 384 poços que já contém siRNA. Centrifugar a placa a 100 g durante 15 segundos a temperatura ambiente. </li><li> Incubar a placa durante 30 minutos à temperatura ambiente. </li><li> Células prontas ensaio em placa, como descrito acima (Seção 2). Centrifugar a placa a 100 g durante 15 segundos a temperatura ambiente. </li><li> Retornar placa de ensaio para incubadora humidificada a 37 ° (5% CO 2) C durante 48-96 horas, tal como determinado para o gene alvo derrubar (ver discussão). </li></ol> 4. Pequeno Screening Molecule <ol><li> Dilui-se o fármaco de interesse (por exemplo, inibidores. Moléculas pequenas) em Opti-MEM a 6x a concentração final desejada. Diluições em série pode ser concluída usando pipetas de mão ou uma estação de tratamento de líquido. </li><li> Adicionar 2,5 mL / poço de composto de teste ou tampão contendo veículo (por exemplo, DMSO) para o lado das cavidades com uma pipeta multicanal. </li><li> Centrifugar a placa a 100 g durante 15 segundos a temperatura ambiente (incubação é opcional). </li></ol> 5. Persistente tratamento com o agonista <ol><li> Prepara-se uma de 600 nm (ou seja. 6 vezes a concentração final desejada de 100 nM), solução de quinpirole em Opti-MEM. </li><li> Adicionar 2,5 mL / poço de solução quinpirole 600 nM para o lado das cavidades com uma pipeta multicanal. </li><li> Centrifugar a placa a 100 g durante 15 segundos a temperatura ambiente, e incubar a 37 ° C numa incubadora humidificada com 5% de CO 2 durante 2 horas. </li></ol> 6. Estimulação de AMPc Acumulação <ol><li> Durante a incubação de 2 horas, preparar as stimulatiem solução em Opti-MEM. A solução de estimulação é composta por 40 uM de forscolina, 2 uM de 3-isobutil-1-methyxanthine (IBMX), e 4 uM espiperona (todas as concentrações no passo 6 são 4x a concentração final desejada). </li><li> Adicionar 5 uL / ​​poço da solução de estimulação para o lado das cavidades com uma pipeta multicanal. </li><li> Centrifugar a placa a 100 g durante 15 segundos a temperatura ambiente, e incubar à temperatura ambiente durante 1 hora. </li></ol> 7. Têmpera e Estimativa de cAMP Acumulação <ol><li> Produção de AMPc pelas células é medida utilizando o kit de cAMP dinâmica 2, de acordo com as instruções do fabricante. Resumidamente, reconstituir anti-cAMP-criptato e cAMP-d2 em água destilada. Congelar alíquotas a -20 ° C para armazenagem de curta duração. </li><li> Dilui-se uma alíquota de anti-AMPc-criptato e uma alíquota de cAMP-D2 separadamente em tampão de lise de acordo com as instruções do fabricante para fazer soluções de trabalho. </li><li> Add 10 ul / poço de solução de trabalho de anti-cAMP-criptato e 10 ul / poço de solução de trabalho de cAMP-D2 para a placa de 384 poços com uma pipeta multicanal. </li><li> Centrifugar a placa a 100 g durante 15 segundos a temperatura ambiente, e incubar à temperatura ambiente durante 1 hora. </li><li> Leia a placa em um leitor de placas de fluorescência (configuração de escala automática para a sensibilidade) com uma excitação de 337 nm e medir as emissões em 620 nm e 665 nm por instruções do fabricante. </li><li> Aplicar análise raciométrica para avaliar curva padrão cAMP por instruções do fabricante. Usando os valores resultantes, extrapolar a acumulação de AMPc estimada nos poços de teste, utilizando o software de análise de dados. </li></ol>

<ol>
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Em um esforço para facilitar os estudos de sensibilização heteróloga, temos extensivamente modificado nosso método anterior alcançar uma simplificada &quot;mix e ler&quot; formato que pode ser alterada a seleção de alto rendimento e mecanismo de exame ação. As modificações importantes nosso protocolo pode ser resumido como se segue: 1) o uso de células criopreservadas como reagentes de ensaio &quot;pronto&quot;, 2) a miniaturização do ensaio em formato de 384 poços, e 3) a utilização da medição de HT...

Um adenililciclase adaptativo (AC) de sinalização de resposta conhecida como a sensibilização de super-heteróloga ou foi descoberto pela primeira vez no laboratório do Prêmio Nobel, Dr. Marshall Nirenberg. Dr. Nirenberg proposto que o aumento da capacidade de resposta AC observado após a ativação do receptor opióide δ crônica era um mecanismo envolvido na tolerância e dependência de opiáceos 1. Além disso a activação do receptor opióide δ crónica, esta resposta neuroadaptativo de sinalização AC também ocorre a seguir a activação persistente de vários outros Gαi / acoplada-o-receptores 2. Notavelmente, muitos destes receptores estão associados com a dor, distúrbios neuropsiquiátricos e neurológicas, e incluem opióide μ / κ, D 2/4 da dopamina, 5HT 1A, e M 2/4 receptores muscarínicos 2. Além de descobertas do Dr. Nirenberg, um grande corpo de evidências que liguem sensibilização de sinalização AC para a ativação do receptor opióide crônica tanto <em&gt; in vitro e in vivo 3-7. Sensibilização de CA também tem sido associado com uma variedade de doenças que envolvem receptores de dopamina D-2, como incluindo esquizofrenia e da doença de Parkinson (para revisão ver referência 2). Apesar da importância potencial de sensibilização, o mecanismo preciso (s) associado com a ativação persistente Gαi / acoplada-o receptor que leva ao aumento da capacidade de resposta AC permanece em grande parte desconhecido. Estes estudos fornecem a justificativa para examinar os mecanismos de sensibilização dos adenililciclase como um importante alvo neurobiológica. Da mesma forma, a relevância fisiológica da AC sinalização 8 e a importância que as isoformas individuais de corrente alternada conter nesta resposta adaptativa também deve ser reconhecido 2,9,10. No contexto de nossa pesquisa, as características gerais associadas com a sensibilização heteróloga das isoformas recombinantes de AC paralelo essas características descrita para estudar as isoformas endógenos de AC. Especificamente, pesquisas anteriores descobriram que a ativação de proteínas Gαi / o e subunidades posterior liberação / βγ rearranjo são requisitos importantes para a sensibilização do receptor induzida de todas as isoformas de AC. Além disso, diversos estudos sugerem que a sinalização de proteina-quinases e subunidades Gβγ esteja envolvido na sensibilização 2,11-13. ACs individuais também exibir padrões de sensibilização únicas e distintas 12. Por exemplo, a exposição persistente de receptores D 2 de agonistas está relacionado com a sensibilização de AC1 e AC8 em Ca 2 + / calmodulina 14,15 estimulação, ao passo que a AC3 estreitamente relacionada não é sensibilizado 2. AC2, AC4, e AC7 estão intimamente relacionados, no entanto, apenas a actividade da PKC-AC2 é estimulado de forma robusta sensibilizadas depois de exposição prolongada de receptores D 2 de agonistas 7,14,16,17. Além disso, AC5 e AC6 mostram um acentuado grau de hetersensibilização para ologous e Gás-acumulação de AMPc estimulada por forskolin após a ativação de receptores D 2 14,18-20, mas parecem diferir em sua exigência para Gβγ subunidade-AC interações 21. Embora a maioria dos estudos de sensibilização AC usaram linhagens de células modelo (por exemplo, células HEK293 expressando isoformas AC individuais), parece que estes resultados traduzem-se em modelos de células neuronais nativos 4,22. Mais recentemente, os efeitos de inibidores selectivos de pequenas molécula de isoformas AC identificados em células HEK293 que expressam as isoformas AC também podem ser convertidos para os estudos in vivo do comportamento 23. A falta de um mecanismo molecular para a identificação de sensibilização heterólogo provavelmente reflecte a complexidade da resposta adaptativa, bem como as propriedades reguladoras únicas do indivíduo AC isoformas 12. Desvendando tal complexidade é ainda mais complicada pelo uso de metodologia pesado tchapéu limitou investigadores académicos de empregar abordagens imparcial. Por exemplo, os nossos estudos mecanísticos envolveu o uso de modelos celulares continuamente cultivadas utilizando 24 - e 48-formato de poço de cultura de tecidos de 15. As células em cultura foram normalmente cultivadas durante 48 horas e, em seguida, submetido a tratamento com drogas agonistas (2-18 horas), seguido por uma série de lavagens e incubações de células (Figura 1). AC-isoforma específica de cAMP de acumulação protocolos foram então empregues, seguido por medição da acumulação de cAMP utilizando um processo trabalhoso e demorado [3 H] metodologia 15,24 ligação cAMP. A duração do início ao fim, para cada ensaio, geralmente requerido um total de quatro a cinco dias de plaqueamento de células para análise de dados (Figura 1). A aplicação de novas tecnologias e automação tem levado a melhorias marcado para estudos de sensibilização no ambiente industrial e HTS centro. Por exemplo, um grupo de trabalho com o Centro Nacional de Chemical Genomics relatou um dia dois HTS processo de ensaio para identificar pequenas moléculas inibidoras da μ do receptor opióide sensibilização induzida no formato 1536-25 bem. O presente artigo descreve os nossos esforços para desenvolver um ensaio HTS para estudos de sensibilização heteróloga utilizando tecnologias que estão disponíveis na maioria das instituições de pesquisa acadêmica. Esta estratégia foi conseguida por incorporação do uso de células criopreservadas de modelos celulares expressando heterologamente o receptor de dopamina D 2 em combinação com as isoformas de adenilil-ciclase recombinante endógenos ou individuais (CHO-D 2L ou células HEK-AC6 / D 2 L). Para melhorar o nosso rendimento, redesenhamos nosso ensaio de sensibilização 48 bem (cerca de&gt; 20 passos mais 4-5 dias) para um de cinco etapas, ensaio dia único em formato de 384 poços que era essencialmente &quot;misturar e ler&quot;. O novo formato utiliza um tempo homogêneo de fluorescência resolvida disponível comercialmente (HTRF) ensaio para medir cAMP accumulation em células intactas com um leitor de placa de multi modo. O ensaio é robusto e passíveis de triagem pequena molécula, e pode ser efectivamente aplicado a triagem de inibidores de sensibilização heteróloga. Além disso, podemos fornecer dados que permitem o uso deste ensaio com transfecção reversa de siRNA para triagem ampla biblioteca siRNA alvo ou genoma, com apenas uma pequena modificação para a abordagem geral.

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		<tr height="28">
			<td height="28" style="height:28px;width:205px;">CHO-K1-DRD2L cells</td>
			<td style="width:144px;">DiscoveRx</td>
			<td style="width:148px;">930579C2</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Ham&#8217;s F12 media</td>
			<td style="width:144px;">VWR</td>
			<td style="width:148px;">SH30026fs</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Fetal Bovine Serum</td>
			<td style="width:144px;">VWR</td>
			<td style="width:148px;">SH3007003</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">G418</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">A1720</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Hygromycin</td>
			<td style="width:144px;">Fisher</td>
			<td style="width:148px;">50-230-5556</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Penicillin/ streptomycin</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">15070063</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">15 cm dishes</td>
			<td style="width:144px;">BD Falcon</td>
			<td style="width:148px;">353025</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">DMSO</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">D2650</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Cell dissociation buffer</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">13150016</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Phosphate Buffered Saline</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">10010-049</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Cryovials</td>
			<td style="width:144px;">Fisher</td>
			<td style="width:148px;">976171</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Opti-MEM</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">31985070</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">TC treated 384 well plates</td>
			<td style="width:144px;">Perkin-Elmer</td>
			<td style="width:148px;">6007688</td>
			<td></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:205px;">HTRF cAMP Dynamic 2 kit</td>
			<td style="width:144px;">Cisbio Bioassays</td>
			<td style="width:148px;">62AM4PEB</td>
			<td style="width:199px;">http://www.htrf.com/camp-cell-based-assay</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Synergy 4</td>
			<td style="width:144px;">Biotek</td>
			<td style="width:148px;">H4MLFPTAD</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Prism 6</td>
			<td style="width:144px;">GraphPad Software</td>
			<td style="width:148px;">NA</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">3-Isobutyl-1-methylxanthine</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">I5879</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">(&#177;)Quinpirole</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">Q111</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Spiperone</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">S7395</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Clozapine</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">C6305</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Haloperidol</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">H1512</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">S-(-)Sulpiride</td>
			<td style="width:144px;">Sigma</td>
			<td style="width:148px;">S112</td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:205px;">Lipofectamine2000 transfection reagent</td>
			<td style="width:144px;">Invitrogen</td>
			<td style="width:148px;">11668019</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Trypan blue</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:148px;">T10282</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Water, DNase, RNase-free</td>
			<td style="width:144px;">MP Biomedicals</td>
			<td style="width:148px;">821739</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Gas siRNA</td>
			<td style="width:144px;">Dharmacon</td>
			<td style="width:148px;">custom siRNA</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">Non-targeting siRNA control</td>
			<td style="width:144px;">Dharmacon</td>
			<td style="width:148px;">D-001206-14-20</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:205px;">siGlo Red</td>
			<td style="width:144px;">Dharmacon</td>
			<td style="width:148px;">D-001630-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

drug-induced sensitization of adenylyl cyclase: assay streamlining and miniaturization for small molecule and sirna screening applications

Sensibilização de adenilil-ciclase (AC) de sinalização tem sido implicada numa variedade de distúrbios neuropsiquiátricos e neurológicas, incluindo o abuso de substâncias e na doença de Parkinson. Activação aguda de Gαi / O-ligada receptores inibe a actividade de AC, enquanto que a activação persistente de estes receptores resulta na sensibilização heteróloga de CA e um aumento dos níveis de AMPc intracelular. Estudos anteriores demonstraram que esta melhoria da capacidade de resposta de CA é observada tanto in vitro e in vivo a seguir a activação crónica de vários tipos de receptores Gαi / ligada a O, incluindo D 2 da dopamina e μ receptores opióides. Apesar de sensibilização heteróloga do AC foi relatada pela primeira vez há quatro décadas, o mecanismo (s) que estão na base deste fenômeno são ainda desconhecidas. A falta de dados mecanicistas presumivelmente reflete a complexidade envolvida com esta resposta adaptativa, o que sugere que as abordagens nonbiased poderia ajudar na identificaçãoção das vias moleculares envolvidas na sensibilização heteróloga do AC. Estudos anteriores têm implicado quinase e sinalização Gbγ como sobreposição de componentes que regulam a sensibilização heteróloga do AC. Para identificar alvos sobrepostos únicos e adicionais associados com a sensibilização da AC, o desenvolvimento ea validação de um ensaio de sensibilização cAMP escalável é necessário para uma maior taxa de transferência. As abordagens anteriores para estudar sensibilização são geralmente complicado que implica a manutenção de cultura de células contínuo, bem como uma complexa metodologia para medição da acumulação de AMPc que envolve vários passos de lavagem. Assim, o desenvolvimento de um ensaio de base celular robusto, que pode ser utilizado para rastreio de alto rendimento (HTS) em um formato de 384 poços iria facilitar estudos futuros. Usando dois modelos celulares D 2 receptores de dopamina (ie. CHO-D 2D e HEK-AC6 / D 2D), convertemos o nosso ensaio de sensibilização 48 poços (&gt; 20 passos 4-5 dias) para um período de cinco step, ensaio dia único em formato de 384 poços. Este novo formato é passível de triagem pequena molécula, e que demonstram que este desenho do ensaio também pode ser facilmente utilizado para a transfecção reversa de siRNA em antecipação de rastreio da biblioteca de siRNA alvo.

Parte I. Desenvolvimento de um ensaio de sensibilização bem heterólogo 384 para a identificação de inibidores de moléculas pequenas utilizando um modelo de células comercialmente disponível. Estudar sensibilização heteróloga em um modelo de celular, fizemos uma série de melhorias que nos permitiu agilizar o ensaio em um formato &quot;misturar e ler&quot;. Várias das modificações principais são destacadas a seguir, e são descritos em mais pormenor na discuss?...

Watch this Scientific Journal Video about Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications at JoVE.com

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Ativação persistente de inibição G receptores acoplados à proteína resulta em sensibilização da adenilil ciclase sinalização. Para identificar as vias moleculares essenciais, abordagens nonbiased são necessárias, no entanto, esta estratégia requer o desenvolvimento de um ensaio escalável sensibilização AMPc baseado em células. Relata-se um ensaio de sensibilização para a pequena molécula e triagem siRNA.

Induzida por drogas Sensibilização de adenililciclase: Racionalização Ensaio e miniaturização para pequenas moléculas e siRNA Triagem Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

drug-induced-sensitization-adenylyl-cyclase-assay-streamlining

Research

JoVE Journal

Bioengineering

12.6K visualizações.  Purdue University.  Ativação persistente de inibição G receptores acoplados à proteína resulta em sensibilização da adenilil ciclase sinalização. Para identificar as vias moleculares essenciais, abordagens nonbiased são necessárias, no entanto, esta estratégia requer o desenvolvimento de um ensaio escalável sensibilização AMPc baseado em células. Relata-se um ensaio de sensibilização para a pequena molécula e triagem siRNA. 

Vídeo: Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

Baseados em células de Ensaio de cálcio para Médio a High Throughput Screening de Funções TRP usando FlexStation Canal 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

Bioengenharia

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

Wideband Detector óptico de ultra-som para aplicações Medical Imaging

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Porous Silicon micropartículas para entrega de siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

Uma rápida e quantitativa método fluorimétrico para-Targeting proteína pequena molécula Rastreio de drogas

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Fabricação de Mecanicamente Tunable e Bioativos metal andaimes para aplicações biomédicas

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formação Light-mediada e padronização de hidrogéis para aplicações de cultura celular

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

Cápsulas à base de plantas de extração de para aplicações de microencapsulação

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

Aplicações para painéis de iluminação compatíveis com microplate de código aberto

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...

Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various biological activities including cell proliferation, survival, migration and differentiation. Several aberrations in the FGFR signaling pathway, owing to mutations or gene amplification events, have been identified in various types of cancers. Hence, recent research has focused on developing strategies involving therapeutic targeting of FGFRs. Current FGFR inhibitors at various stages of pre-clinical and clinical development include either small molecule inhibitors of tyrosine kinases or monoclonal antibodies, with only a few peptide- based inhibitors in the pipeline. Here, we provide a protocol using phage display technology to screen small peptides as antagonists of FGFR2. Briefly, a library of phage-displayed peptides was incubated in a plate coated with FGFR2. Subsequently, unbound phage was washed off by TBST (TBS + 0.1% [v/v] Tween-20), and bound phage was eluted with 0.2 M glycine-HCl buffer (pH 2.2). The eluted phage was further amplified and used as input for the next round of biopanning. Following three rounds of biopanning, the peptide sequences of individual phage clones were identified by DNA sequencing. Finally, the screened peptides were synthesized and analyzed for affinity and biological activity.

Herein, we present a detailed protocol for screening small peptides that bind to FGFR2 using a phage display peptide library. We further analyze the affinity of the selected peptides toward FGFR2 in vitro and its ability to suppress cell proliferation.

Triagem e identificação de pequenos peptídeos visando o fator de crescimento de fibroblastos Receptor2 usando uma biblioteca de peptídeos de Phage display

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various ...

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.

Here we present a protocol to design and fabricate custom microfluidic devices with minimal financial and time investment. The aim is to facilitate the adoption of microfluidic technologies in biomedical research laboratories and educational settings.