Agradecemos ao professor Lobo Frommer (Carnegie Institution for Science, da Universidade de Stanford) para gentilmente nos fornecer a glicose FRET plasmídeo e Dr. Uwe Ludewig (Universidade Hohenheim) por gentilmente fornecer a glutamina FRET construir no pUTKan planta vetor de expressão. AB é financiado pelo programa Studentships Prioritárias BBSRC alvejado. Ambos CK e KP são suportados por RCUK Bolsas em Processamento de Biofármacos. CK também agradece a Lonza Biologics pelo apoio financeiro. O Centro de Biologia Sintética e Inovação é generosamente apoiado pelo EPSRC.

1. Celular Linha Revival e Manutenção <ol><li> Reviver as células CHO em 9 ml de meio CD-CHO suplementado com 8 mM de L-glutamina e 10 ml / L de suplemento de 100x hipoxantina / timidina (meio de crescimento completo). </li><li> Centrifugar a 100 xg durante 5 min. </li><li> Ressuspender as células em 10 ml de meio de crescimento completo fresco. </li><li> Retirar uma amostra de 1 ml e determinar a concentração de células viáveis ​​por microscopia de luz, utilizando o método de exclusão do corante azul tripano num hemocitómetro. </li><li> Iniciar uma cultura com uma densidade de sementeira de 3 x 10 5 células / ml em 125 ml de frascos de agitação. </li><li> Manter a cultura numa incubadora a 37 ° C, numa atmosfera humidificada de 5% de CO 2, numa plataforma de agitação orbital rotativo a 120 rpm. </li><li> Subcultura a cada 3-4 dias em meio de crescimento completo com uma densidade de sementeira de 2 x 10 5 células / ml. </li></ol> 2. A transfecção da linha celular com o plasmídeo Containing o Biosensor Gene <ol><li> Preparar DNA de plasmídeo utilizando um kit de purificação de plasmídeo em larga escala. </li><li> Manter as células em condições adequadas (37 ° C, 5% CO 2) 12-24 h antes da transfecção para assegurar que as células são activamente dividir no tempo de transfecção. </li><li> Contar as células usando o método de exclusão do corante azul de tripano, em seguida, se preparar um volume de trabalho de 20 ml de cultura de células em um frasco de agitação de 125 ml a uma concentração de 1 x 10 6 células / ml. </li><li> Usar um kit de transfecção adequado para a linha celular em uso. O DNA de plasmídeo a proporção de reagentes de transfecção vai ser dependente da linha de células e o vector. Recomenda-se que esta é otimizado pelo teste de uma gama de relações dentro do intervalo especificado pelo fabricante antes de realizar as transfections finais. Também manter, pelo menos, uma cultura de controlo negativo que contém as células, mas não o ADN. </li><li> Incubar durante 4 dias em modo estático e, em seguida, transferir para um Shaking plataforma rotativa a 125 rpm. </li><li> Adicionar o antibiótico apropriado para a selecção do plasmídeo a uma concentração apropriada, tal como determinado por uma curva de mortalidade. Neste estudo, zeocina foi adicionado a uma concentração final de 400 ug / ml para selecção de células transfectadas. </li><li> Mudança de mídia em um intervalo de tempo adequado para a linha celular, acrescentando antibiótico a cada vez, até que as células em controle de poço morreram. </li><li> Use os poços mais confluentes para avançar para agitando culturas. </li><li> Estabelecer um banco de células, congelando as células em frascos criogénicos contendo 10 7 células viáveis ​​em 1 ml da mistura de congelação. Neste trabalho, esta é constituída por 92,5% de meio de crescimento e de 7,5% de dimetil sulfóxido. </li></ol> 3. Batch e Fed-batch Crescimento Celular Curve <ol><li> Seguir as instruções para a manutenção das células acima para estabelecer culturas de células em triplicado de células CHO transfectadas em 250 ml de frascos de agitação com um volume de trabalho de 50 ml. </li><li> Manter a cultures em um incubador humidificado de células a 37 ° C, com 5% de CO 2, numa plataforma de agitação orbital rotativo a 125 rpm, e retirar amostras de 4,1 ml a partir de cada cultura de crescimento a intervalos de 24 h. </li><li> Utilizar 100 ul da amostra para determinar a concentração de células e viabilidade celular viável com o método de exclusão de corante azul de tripano. </li><li> Repetir até que a concentração de células viáveis ​​é reduzido a zero. </li><li> Repetir para as culturas de células alimentado com alimentação suplementar com a quantidade apropriada de glicose ou glutamina no dia 6 para restaurar as suas concentrações para os seus valores iniciais de 36 mM e 4 mM, respectivamente. Recomenda-se que as culturas de controlo adicional, também são mantidos, que estão a ser alimentados com o mesmo volume (12 ml, no caso) de água pura. </li></ol> 4. FRET Medidas Relação <ol><li> Tome 2 ml das amostras diárias removidos na etapa 3.2 e centrifugar a 100 xg durante 5 min a 4 ° C. </li><li> Resuspterminou o sedimento celular em 2 ml de gelo frio de fosfato salina tamponada (PBS). Transferir este em uma placa de 6 poços e adicionar uma amostra de branco não contendo células para um poço. </li><li> Medir os níveis de fluorescência azul e amarelo imediatamente em um comprimento de onda de excitação de 430/435 nm e emissão de comprimentos de onda 465/435 nm (azul) e 520/510 nm (amarelo). </li><li> Calcular as relações de FRET (razão de fluorescência amarela detectados mais de fluorescência azul detectado). </li></ol> 5. Metabolite Ensaios <ol><li> Tome 2 ml das amostras diárias removidos na etapa 3.2 e centrifugar a 100 xg durante 5 min a 4 ° C. Remover o sobrenadante. </li><li> Ressuspender o sedimento de células em 3 ml de PBS gelado e centrifugar novamente a 100 x g durante 5 min. </li><li> Remover o sobrenadante e ressuspender o sedimento de células em 3 ml de PBS gelado, como acima. Sonicar o 5x amostra por 3 min cada um em um pulso de 15 segundos ligado e 15 segundos desligado. Neste ponto, a célula adicionalcts podem ser congeladas, se desejar. </li><li> Utilizado um kit de ensaio de glucose apropriada conforme as instruções do fabricante, em amostras de extractos de células para determinar a concentração de glucose intracelular (ver tabela de materiais). Nota: Use padrões apropriados para a construção de uma curva padrão. Neste trabalho, a concentração dos padrões variaram entre 0-100 mM. </li><li> Usando as leituras para os padrões, construir a curva padrão e encontrar a equação linear de melhor ajuste (neste caso: y = 1310.51x - 723,43, onde y é a leitura de absorvância e x é a concentração de glucose). </li><li> Calcular as concentrações de glicose utilizando a curva padrão e tendo em conta a diluição. A concentração de glucose intracelular pode ser calculada utilizando a seguinte equação: <img alt="" fo:content-width="4.5in" src="/files/ftp_upload/51200/book_bg.png" width="500" /> Para as células CHO, o volume da célula única foi calculada usando um diâmetro de célula de 12 pM 12 </sup&gt;. </li><li> Usar um kit de ensaio de glutamina apropriado conforme as instruções do fabricante, em amostras de extractos de células para determinar a concentração de glutamina intracelular (ver tabela de materiais). Nota: Use padrões apropriados para a construção de uma curva padrão. Neste trabalho, a concentração dos padrões variaram entre 0-2 mM. </li><li> Como em 5.5 acima, a curva de calibração (neste caso, y = 203.1x + 17.1, onde y é a leitura de absorvância e x é a concentração de glucose) </li><li> Usando a curva padrão, calcular a concentração de glutamina das amostras, tendo em conta a diluição. A concentração de glutamina intracelular pode ser calculado usando a Equação 1 acima. </li></ol>

<ol>
	<li>Moser, I., Jobst, G., Urban, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosensor+arrays+for+simultaneous+measurement+of+glucose,+lactate,+glutamate,+and+glutamine.">Biosensor arrays for simultaneous measurement of glucose, lactate, glutamate, and glutamine.</a> Biosens. Bioelectron. 17 (4), 297-302 (2002).</li><li>Billingsley, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+nano-optodes+for+glucose+detection.">Fluorescent nano-optodes for glucose detection.</a> Anal. Chem. 82 (9), 3707-3713 (2010).</li><li>Dattelbaum, J. D., Lakowicz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Determination+of+Glutamine+Using+a+Genetically+Engineered+Protein.">Optical Determination of Glutamine Using a Genetically Engineered Protein.</a> Anal. Biochem. 291 (1), 88-95 (2001).</li><li>Kfouri, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+miniaturized+wireless+fluorescence-based+diagnostic+imaging+system.">Toward a miniaturized wireless fluorescence-based diagnostic imaging system.</a> IEEE J. Sel. Top. Quantum Electron. 14 (1), 226-234 (2008).</li><li>Dittrich, P. S., Manz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+fluorescence+detection+in+microfluidic+channels&#8212;the+Holy+Grail+in+muTAS.">Single-molecule fluorescence detection in microfluidic channels&#8212;the Holy Grail in muTAS.</a> Analytical and Bioanalytical Chemistry. 382 (8), 1771-1782 (2005).</li><li>Hou, B. H., Codamo, J., Pilbrough, W., Hughes, B., Gray, P. P., Munro, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+sensors+for+monitoring+dynamic+changes+of+intracellular+metabolite+levels+in+mammalian+cells.">Optical sensors for monitoring dynamic changes of intracellular metabolite levels in mammalian cells.</a> Nat. Protoc. 6 (7), 1818-1833 (2011).</li><li>Bermejo, C., Haerizadeh, F., Takanaga, H., Chermak, D., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+cytosolic+glucose+and+ATP+levels+in+yeast+using+optical+sensors.">Dynamic analysis of cytosolic glucose and ATP levels in yeast using optical sensors.</a> Biochem. J. 432, 399-406 (2010).</li><li>Yang, H. Y., Bogner, M., Stierhof, Y. D., Ludewig, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H(+)-independent+glutamine+transport+in+plant+root+tips.">H(+)-independent glutamine transport in plant root tips.</a> Plos One. 5, (2010).</li><li>Fehr, M., Takanaga, H., Ehrhardt, D. W., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+high-cavacity+bidirectional+glucose+transport+across+the+endoplasmic+reticulum+membrane+by+genetically+encoded+fluorescence+resonance+energy+transfer+nanosensors.">Evidence for high-cavacity bidirectional glucose transport across the endoplasmic reticulum membrane by genetically encoded fluorescence resonance energy transfer nanosensors.</a> Mol. Cell. Biol. 25 (24), 11102-11112 (2005).</li><li>Ai, H. W., Hazelwood, K. L., Davidson, M. W., Campbell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+protein+FRET+pairs+for+ratiometric+imaging+of+dual+biosensors.">Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors.</a> Nat. Methods. 5 (5), 401-403 (2008).</li><li>Ouyang, M. X., Sun, J., Chien, S., Wang, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+hierarchical+relationship+of+Src+and+Rac+at+subcellular+locations+with+FRET+biosensors.">Determination of hierarchical relationship of Src and Rac at subcellular locations with FRET biosensors.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (38), 14353-14358 (2008).</li><li>Han, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+recombinant+Chinese+hamster+ovary+cells+grown+as+suspended+aggregates+in+stirred+vessels.">Cultivation of recombinant Chinese hamster ovary cells grown as suspended aggregates in stirred vessels.</a> J. Biosci. Bioeng. 102 (5), 430-435 (2006).</li><li>Deuschle, K., Okumoto, S., Fehr, M., Looger, L. L., Kozhukh, L., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+optimization+of+a+family+of+genetically+encoded+metabolite+sensors+by+semirational+protein+engineering.">Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering.</a> Protein Sci. 14 (9), 2304-2314 (2005).</li><li>Wong, D. C. F., Wong, N. S. C., Goh, J. S. Y., May, L. M., Yap, M. G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+N-Glycosylation+gene+expression+in+CHO+cell+Fed-batch+cultures.">Profiling of N-Glycosylation gene expression in CHO cell Fed-batch cultures.</a> Biotechnol. Bioeng. 107 (2), 516-528 (2010).</li><li>Behjousiar, A., Kontoravdi, C., Polizzi, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Situ+Monitoring+of+Intracellular+Glucose+and+Glutamine+in+CHO+Cell+Culture.">In Situ Monitoring of Intracellular Glucose and Glutamine in CHO Cell Culture.</a> PLoS One. 7, (2012).</li><li>Dwyer, M. A., Hellinga, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periplasmic+binding+proteins:+a+versatile+superfamily+for+protein+engineering.">Periplasmic binding proteins: a versatile superfamily for protein engineering.</a> Curr. Opin. Struct. Biol. 14 (4), 495-504 (2004).</li></ol>

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Os FIBS activar in vivo e in situ, a quantificação de moléculas-chave, neste caso nutrientes limitantes do crescimento, eliminando incertezas decorrentes de têmpera e métodos de extracção. Os resultados sugerem que existe uma boa correlação entre o sinal FIBS e as concentrações intracelulares na gama de 1-5 mM de glicose e 0,3-2 mM de glutamina. No lote CHO cultura de células, estas concentrações são encontradas nas fases de declínio exponencial, estacionárias, e no início. As fases exponencia...

Monitoramento metabolito tem diversas aplicações em bioprocessing, incluindo o desenvolvimento de processos, mídia e design de alimentação e engenharia metabólica. Vários métodos estão disponíveis para as medições de concentração através da utilização de um enzimática, química 2, ou ensaios de ligação 3. Uma opção interessante é a utilização de fluorescência, em sensores biológicos in vivo como repórteres da concentração intracelular de metabolitos essenciais à base de proteínas. Em ensaios de ultra-baixo volume, a fluorescência é uma ferramenta conveniente como miniaturização realmente melhora a taxa de 4,5 sinal-ruído e sensores à base de proteínas podem ser geneticamente codificados sentido de que não há reagentes exógenos são necessários para análise de metabolitos. Transferência de Energia de Ressonância de Förster (FRET) biossensores consistem de duas proteínas fluorescentes ligadas por um domínio de ligação ao ligando. FRET é uma transferência não radiativa de energia a partir de um doador de foto-animado para um receptor molécula fluorescente loca<html> ed em estreita proximidade (&lt;100). Clivagem ligando ou ligação provoca uma alteração conformacional no sensor, o qual, por sua vez, induz uma mudança na proximidade dos fluoróforos, conduzindo a uma alteração na eficiência de TERF medido pela alteração no espectro de emissão. Sensores biológicos FRET integrados (FIBS) são constitutivamente produzido dentro da linha de células e as suas características espectrais são alteradas com base na concentração de metabolito no interior da célula. FIBS têm tempos de resposta rápidos, tornando-os ideais para a tomada de medidas para os modelos dinâmicos 6. Aplicações anteriores incluem o monitoramento único 7-9 e múltiplas 10 metabólitos e fornecendo dados sobre a distribuição espaço-temporal 11. FIBS podem ser criados de duas configurações: Apo-Max, onde a ligação do ligando perturba a proximidade dos fluoróforos reduzindo a transferência de energia e de Apo-Min, onde a ligação do ligando traz os dois fluoróforos em contacto mais próximo (Figura 1). _content &quot;&gt; Neste trabalho, um protocolo para a construção são apresentados de ovário de hamster chinês (CHO), linhas celulares que expressam constitutivamente um FIBS para um metabolito de glucose ou glutamina. está estabelecido um método para a calibração do sensor in vivo, para permitir futura quantitativa medições de concentração de metabolito intracelular, tal como apresentado na Figura 2. Baseado nisto, a concentração intracelular de glucose ou glutamina pode ser determinada em fed-batch culturas de células CHO, em que os dois nutrientes foram adicionados em concentrações elevadas no processo. Os resultados demonstram que a utilização do sinal de fluorescência a partir das FIBS e da curva de calibração previamente construído, com precisão de previsão da concentração intracelular é possível, tal como foi confirmado por um ensaio enzimático independente. Este método oferece vantagens substanciais em relação às tecnologias analíticas correntes, porque ele não é invasivo, de baixo custo e rápida, dando um sinal em tempo real dos FIBS que podem ser monitored em toda a cultura.

<table border="0" cellpadding="0" cellspacing="0" width="936">
	<colgroup>
		<col />
		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">CHO-S Cells</td>
			<td style="width:120px;">Life Tecnologies</td>
			<td style="width:120px;">11619-012</td>
			<td style="width:459px;">Cell line will vary depending on the goals of the study</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">pCDNA4/TO vector&#160;</td>
			<td style="width:120px;">Life Tecnologies</td>
			<td style="width:120px;"></td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">TransIT-PRO Transfection Reagent</td>
			<td style="width:120px;">Mirus Bio</td>
			<td style="width:120px;">MIR 5700</td>
			<td style="width:459px;">Transfections can be accomplished using any method suitable for the cell line under study</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Zeocin</td>
			<td style="width:120px;">Invivogen</td>
			<td style="width:120px;"></td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">CD-CHO medium</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">10743-011</td>
			<td style="width:459px;">Cell growth medium is dependent upon the cells under study</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">100X HT Supplement</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">11067-030</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">L-Glutamine 200 mM (100X), Liquid</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">25030032</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">InfinitePRO 200 plate reader</td>
			<td style="width:120px;">Tecan</td>
			<td style="width:120px;">FLx800TBI</td>
			<td style="width:459px;">Any 96-well fluorescence plate reader that can access the required wavelengths can be substituted</td>
		</tr>
		<tr height="21">
			<td height="190" rowspan="6" style="height:190px;width:239px;">Filters for plate reader</td>
			<td rowspan="6" style="width:120px;">Tecan</td>
			<td align="right" style="width:120px;">30000463</td>
			<td rowspan="6" style="width:459px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">(520NM BW 10NM)</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;width:120px;">30022786</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">(430NM BW 35NM)</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;width:120px;">30022787</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:120px;">(465NM BW 35NM)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Maxiprep Plasmid Purification Kit</td>
			<td style="width:120px;">Qiagen</td>
			<td style="width:120px;">12163</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Amplex Red glucose/glucose oxidase assay kit</td>
			<td style="width:120px;">Invitrogen</td>
			<td style="width:120px;">A22189</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">EnzyChrom&#160; Glutamine Assay Kit</td>
			<td style="width:120px;">BioAssay systems</td>
			<td style="width:120px;">EOAC-100</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Improved Neubauer haemocytometer</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:120px;">MNK-420-010N</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Incubator&#160;</td>
			<td style="width:120px;">Nuaire</td>
			<td style="width:120px;">NU-5510E</td>
			<td style="width:459px;"></td>
		</tr>
	</tbody>
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fibs-enabled noninvasive metabolic profiling

Na era da biologia computacional, novos sistemas experimentais de alto rendimento são necessárias para preencher e refinar os modelos para que possam ser validadas para fins preditivos. Idealmente esses sistemas seria baixo volume, o que impede análises de amostragem e destrutivas, quando os dados do curso do tempo devem ser obtidos. O que é necessário é uma ferramenta de monitoramento em situ que pode relatar as informações necessárias em tempo real e de forma não invasiva. Uma opção interessante é a utilização de fluorescência, em sensores biológicos in vivo como repórteres de concentrações intracelulares baseados em proteína. Uma classe especial de in vivo biossensores que tem encontrado aplicações em metabolito quantificação baseia-se na transferência de energia de ressonância de Förster (FRET) entre duas proteínas fluorescentes ligadas por um domínio de ligação ao ligando. FRET sensores biológicos integrados (FIBS) são constitutivamente produzido dentro da linha celular, eles têm tempos de resposta rápidos e sua cha espectralcas mudar de acordo com a concentração de metabolito no interior da célula. Neste papel, o método para a construção de ovário de hamster chinês (CHO), linhas celulares que expressam constitutivamente um FIBS para glicose e glutamina e calibrar os FIBS in vivo em cultura de células em lotes a fim de permitir futura quantificação da concentração intracelular do metabolito é descrito. Os dados de batelada alimentada CHO culturas celulares demonstra que os FIBS foi capaz, em cada caso, para detectar a alteração resultante na concentração intracelular. Usando o sinal de fluorescência a partir das FIBS e a curva de calibração construída anteriormente, a concentração intracelular foi determinada com precisão, como confirmado por um ensaio enzimático independente.

Uma visão geral da metodologia é apresentada na Figura 2. No trabalho aqui apresentado, as células CHO foram transfectadas com o vector de linhas de células estáveis ​​foram seleccionadas FIBS e a uma pressão de antibiótico de 400 ug / ml de zeocina. Por conseguinte, foram criadas duas linhas de células estáveis ​​que expressam constitutivamente separados os sensores de glicose e glutamina. Figura 1 representa as configurações dos dois biossensores utilizados neste es...

Watch this Scientific Journal Video about FIBS-enabled Noninvasive Metabolic Profiling at JoVE.com

FIBS-enabled Noninvasive Metabolic Profiling

Uma descrição de como calibrar transferência Förster Resonance Energy sensores biológicos integrados (FIBS) no perfil metabólico situ é apresentado. Os FIBS pode ser utilizado para medir os níveis intracelulares de metabolitos de forma não invasiva, contribuindo para o desenvolvimento de modelos metabólicas e elevado rendimento de rastreio de condições de bioprocessamento.

FIBS habilitado Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

fibs-enabled-noninvasive-metabolic-profiling

Research

JoVE Journal

Bioengineering

9.6K visualizações.  Imperial College London.  Uma descrição de como calibrar transferência Förster Resonance Energy sensores biológicos integrados (FIBS) no perfil metabólico situ é apresentado. Os FIBS pode ser utilizado para medir os níveis intracelulares de metabolitos de forma não invasiva, contribuindo para o desenvolvimento de modelos metabólicas e elevado rendimento de rastreio de condições de bioprocessamento. 

Vídeo: FIBS-enabled Noninvasive Metabolic Profiling

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

Profiling MicroRNA de Alto Rendimento: Multiplex Optimized qRT-PCR em Escala nanolitros no IFCS Fluidigm dinâmico ArrayTM

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

Bioengenharia

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e.&#160;geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.

In situ compressão de carga e de imagem não invasivos Correlative do Osso-Ligamento periodontal de dentes fibrosa Joint

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

Quantificação e Tamanho-profiling de vesículas extracelulares Usando Sintonizável resistiva Pulso Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Fácil e precisa Mecano-profiling em micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing termini of oligo- and polysaccharides, as well as its unique chemical characteristics, sialic acid is involved in a multitude of different receptor-ligand interactions. By modifying the expression of sialic acid on the cell surface, sialic acid-dependent interactions will consequently be influenced. This can be helpful to investigate sialic acid-dependent interactions and has the potential to influence certain diseases in a beneficial way. Via metabolic glycoengineering (MGE), the expression of sialic acid on the cell surface can be modulated. Herein, cells, tissues, or even entire animals are treated with C2-modified derivatives of N-acetylmannosamine (ManNAc). These amino sugars act as sialic acid precursor molecules and therefore are metabolized to the corresponding sialic acid species and expressed on glycoconjugates. Applying this method produces intriguing effects on various biological processes. For example, it can drastically reduce&#160;the expression of polysialic acid (polySia) in treated neuronal cells and thus affects neuronal growth and differentiation. Here, we show the chemical synthesis of two of the most common C2-modified N-acylmannosamine derivatives, N-propionylmannosamine (ManNProp) as well as N-butanoylmannosamine (ManNBut), and further show how these non-natural amino sugars can be applied in cell culture experiments. The expression of modified sialic acid species is quantified by high performance liquid chromatography (HPLC) and further analyzed via mass spectrometry. The effects on polysialic acid expression are elucidated via Western blot using a commercially available polysialic acid antibody.

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

Glycoengineering metabólica do ácido siálico usando N-acil-modificado Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing ...

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic resonance (MR) microscopy data. While it is possible to perform MR collections on living, excised mammalian tissue, such experiments have traditionally been constrained by resolution limits and are thus incapable of visualizing tissue microstructure. Conversely, MR protocols that did achieve microscopic image resolution required the use of fixed samples to accommodate the need for static, unchanging conditions over lengthy scan times. The current protocol describes the first available MR technique that enables imaging of living, mammalian tissue samples at microscopic resolutions. Such data is of great importance to the understanding of how pathology-based contrast changes occurring at the microscopic level influence the content of macroscopic MR scans such as those used in the clinic. Once such an understanding is realized, diagnostic methods with greater sensitivity and accuracy can be developed, which will translate directly to earlier disease treatment, more accurate therapy monitoring and improved patient outcomes.
While the described methodology focuses on brain slice preparations, the protocol is adaptable to any excised tissue slice given that changes are made to the gas and perfusate preparations to accommodate the tissue&#39;s specific metabolic needs. Successful execution of the protocol should result in living, acute slice preparations that exhibit MR diffusion signal stability for periods up to 15.5 h. The primary advantages of the current system over other MR compatible perfusion apparatuses are its compatibility with the MR microscopy hardware required to attain higher resolution images and ability to provide constant, uninterrupted flow with carefully regulated perfusate conditions. Reduced sample throughput is a consideration with this design as only one tissue slice may be imaged at a time.

The current protocol describes a method by which users can maintain viability of acute hippocampal and cortical slice preparations during the collection of magnetic resonance microscopy data.

Suporte metabólico de extirpado, tecidos de cérebro vivos durante a aquisição de microscopia de ressonância magnética

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Calorespirometry: Uma abordagem poderosa, não-invasiva para investigar o metabolismo energético celular

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Nanosensors para detectar Protease atividade In Vivo para diagnóstico não-invasivo

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Medição de metaloproteinase matriz celular Global e atividade metabólica em hidrogel 3D

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Um modelo de cultura de matriz-adipócito extracelular humano para estudar crosstalk metabólico de matriz e células

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...