Este trabalho foi financiado em parte por doações do Sul do Texas Technology Management (POCrr 2009,041), o Instituto para a Integração de Medicina e Ciência do Centro Nacional de Pesquisa de Recursos (UL 1RR025767), e do Instituto Nacional de Pesquisa Dental e Craniofacial ( 5R21DE017294).

1. Preparação de Slides funcionalizados <ol><li> Colocar as lâminas de microscópio em um porta-lâminas removível, e lava-se duas vezes por imersão em um frasco de coloração contendo etanol a 99% (grau histológico). Limpe as lâminas limpar usar toalhas de papel (garantia não gerar poeira de papel) e secos usando um jato de gás comprimido nitrogênio. </li></ol> NOTA: Não use Kim-Wipes para limpar as lâminas como iria gerar poeira de papel fino. <ol start="2"><li> Mergulha-se o porta-lâminas contendo as lâminas em um frasco de coloração com ácido sulfúrico concentrado e incube à temperatura ambiente durante a noite. </li></ol> NOTA: Para evitar o contato com a pele, usar produtos químicos resistentes luvas e óculos de segurança ao trabalhar com ácidos concentrados e produtos químicos tóxicos e corrosivos. <ol start="3"><li> Sonicar as lâminas durante 30 min e lavagem com Milli-Q (18 mohms) de água durante 30 min, seguir com outra lavagem em umcetona durante 5 min. Este tratamento expõe os grupos silanol na superfície do vidro. </li><li> Revestir as lâminas limpas com 2,5% (vol / vol em água) de 3-aminopropiltrietoxissilano (APTES) de solução, por imersão do porta-lâminas em APTES durante 30 min, lavar 3 vezes em água Milli-Q durante 15 min cada lavagem, e panificação as lâminas num forno a 110 ° C durante 15 min. Cozimento permite a ligação cruzada das APTES, resultando em-NH2 funcionalização de superfície. </li></ol> NOTA: Verifique a solução APTES em um recipiente de plástico desde APTES depósitos preferencialmente nas paredes do recipiente de vidro. <ol start="5"><li> Usando um spin revestidor revestimento, as lâminas com 1% (peso / vol em tolueno) de poliestireno-Co-anidrido maleico (PS-MA) (Sigma) para atingir uma mono-camada de revestimento hidrofóbico 6. Adicionar 2,0 mL de PS-MA numa lâmina de vidro limpo montado sobre um revestidor de spin e pelagem a 3.000 rpm durante 30 s. Estas condições podem variar de acordo com parâmetros de rotação de revestimento como revestimento paralução, o substrato de espessura, de revestimento e regulada pela seguinte equação 7 </li></ol><img alt="Equação 1" src="/files/ftp_upload/3845/3845eq1.jpg" /> em que h é a espessura da película de revestimento, e é a taxa de evaporação, η, C e ρ são a concentração de viscosidade e densidade da solução de revestimento, respectivamente, e ω é a velocidade angular. NOTA: O tolueno é prejudicial quando inalado por um longo tempo e da utilização de um exaustor é recomendada. <ol start="6"><li> As lâminas podem ser armazenadas em um porta-lâminas para até um mês em seco, o pó condições isentas de a 2 - 8 ° C. </li></ol> 2. Preparação dos inóculos de leveduras e encapsulamento de colágeno <ol><li> Prepare uma cultura durante a noite de C. albicans estirpe SC5314, em levedura Peptona Dextrose [YPD; 10 g / L de extracto de levedura, 20 g / L de peptona e 20 g / L de dextrose] meio líquido por inoculação de uma única colónia de C. albicans em 10-20 mL de YPD 8. Incubar cultura em um agitador orbital (cerca de 150 - 200 rpm) a 30 ° C durante a noite. Colheita 1 mL de Candida albicans células de leveduras obtidas durante a noite culturas YPD (por centrifugação a 5.000 rpm durante 5-10 minutos) e lava-se duas vezes durante 10 min em 1 ml de fosfato estéril tamponada salina (PBS; tampão fosfato 10 mM, 2,7 mM cloreto de potássio, cloreto de sódio 137 mM, pH 7,4) (Sigma) por lavagem. Colheita lavada células também por centrifugação a 1900 rpm durante 10 minutos. Ressuspender as células lavadas em tampão de 1 a reconstrução mL (0,2 N solução de NaOH com bicarbonato de sódio 2,2% (peso / vol) e 4,8% (p / vol) de HEPES, pH 7,2). </li></ol> NOTA: C. albicans é um microorganismo de Risco 1/BSL1 Grupo. Lembre-se sempre usar boas técnicas assépticas / estéril para o trabalho com este microorganismo e seguir os procedimentos institucionais para o descarte adequado de materiais perigosos. <ol start="2"><li> Contar as células utilizando um hemocytometer sobre um microscópio de campo brilhante e se ajustar a uma densidade celular de 5 x 10 7 células / mL. </li><li> Além disso diluir a suspensão de dez vezes por adição de 10 × RPMI-1640 suplementado com L-glutamina e tamponado com morpholinepropanesulfonic (MOPS) de ácido (pH 7,2). </li><li> Encapsular as células de levedura em colagénio por mistura da suspensão de células em meio RPMI-1640 com colagénio (1,8 mg / mL) (Tipo 1 a partir de cauda de rato, BD Biosciences, Bedford, MA), para se obter uma concentração final de 4 × 10 6 células / mL. Manter a suspensão de colagénio de células em gelo para evitar a gelificação do colagénio antes da impressão. </li></ol> 3. Preparação de Ca BChip <ol><li> Limpar e desinfectar todas as superfícies do microarrayer, incluindo a placa de lavagem estação de origem, e da estação de vácuo, travessa corrediça vácuo e câmara de impressão, limpando-o com 70% de isopropanol. </li><li> Coloque e mantenha a vácuo o número desejado de slides PS-MA revestidos de vidro sobre o conjunto de slides do microfoneroarrayer. </li><li> Vórtice a suspensão da célula em colagénio vigorosamente e aspirar 100 uL da suspensão bem misturada em um poço de uma placa de 96 poços, pouco antes da impressão. Coloque esta placa de origem na estação de carregamento. </li><li> Ligue o humidificador para manter uma humidade relativa de 100% na câmara de microarray ao longo do processo de impressão. </li><li> Imprimir 50 nL de suspensão de células em uma matriz de 48 linhas e 16 colunas com manchas espaçadas de 1,2 mm para além usando um spotter microarray (Omnigrid Micro, Inc. Digilab, Holliston, MA) por não-contato deposição usando cónica cônicos 190 dicas mM orifício de cerâmica (Digilab). </li><li> Primeiro, lavar e aspirar-seque as pontas duas vezes depois de cada rodada de impressão. </li><li> Imediatamente após a impressão, coloque a lâmina em uma câmara estanque ao ar, humedecido (cassete de hibridação, ArrayIt Corporation, Sunnyvale, CA) e incubar a 37 ° C durante 24 horas para permitir a formação de biofilmes. </li></ol> 4. Testes de susceptibilidade de PrBiofilmes eformed em Ca BChip contra agentes antifúngicos <ol><li> De soluções de reserva ou pó de drogas antifúngicas, preparar uma solução de trabalho em meio RPMI-1640. Típicos concentrações máximas da solução de trabalho são 1.024 ug / mL para fluconazol e 16 ug / mL para a anfotericina B 5. Outras concentrações podem ser utilizados para diferentes agentes. </li><li> Preparar oito concentrações diferentes do composto por diluição da solução estoque em duas diluições, abrangendo uma gama com o IC 50 estimado do composto no meio. </li><li> Após 24 h de crescimento do biofilme, utilizar o microarrayer para imprimir 50 nL de oito diferentes concentrações dos fármacos, juntamente com positiva (isto é, não existem células de drogas em meios apenas) e negativo (tratada com hipoclorito de sódio durante 20 min) controlos, em pelo menos seis repetições em cima dos biofilmes. </li></ol> NOTA: Manter uma humidade relativa de 100% para evitar a secagem das manchas, enquanto suplementoção das drogas. <ol start="4"><li> Logo após a adição dos fármacos, incubar o chip com os medicamentos em uma câmara humidificada a 37 ° C durante 24 hr. </li><li> Lavar os medicamentos fora por dunking o CaBChip 3-5 vezes durante 2 min de cada vez em PBS e mancha por incubação da BChip Ca com 0,5 uM fun1 a 37 ° C durante 30 min 9. </li><li> Lava-se a mancha por dunking o BChip Ca 3-5 vezes em PBS durante 1-3 min cada enterrada, e secar a BChip Ca usando uma corrente de azoto. Ler a intensidade de fluorescência utilizando um digitalizador microarray (GenePix 4100A, Axon Instruments, CA) com um comprimento de onda de 532 nm com um ganho de PMT de 380. </li><li> Definir a intensidade de fluorescência do controlo positivo (sem fármaco) e mortos (lixívia) tratados com manchas de biofilme a 100% e 0%, respectivamente. </li><li> Determinar a percentagem de inibição pela redução na intensidade de fluorescência (F) em relação à média dos pontos de controlo utilizando a seguinte equação </li></ol><img alt="Equação 2" src="/files/ftp_upload/3845/3845eq2.jpg" /> onde F, F max e F-primas são o intensidades de fluorescência de drogas tratados, nenhum medicamento de controle e tratados com lixívia pontos, respectivamente. As configurações do scanner foram ajustados para obter F max e F o de 30000 e 4000 RFU, respectivamente. <ol start="9"><li> Calcular a concentração inibitória 50% ou IC 50 encaixando a equação de Hill variável inclinação utilizando GraphPad Software Prism (La Jolla, CA). </li></ol> 5. Os resultados representativos Um representante Ca BChip, consistindo de um 48 × 16 conjunto de nano-biofilmes de C. albicans, é mostrado na Figura 2. A microscopia de campo claro mostra uma característica geral de arquitetura do nano-biofilme. As imagens microscópicas de elétrons de digitalização do show que o biofilme hifas fúngicas são incorporados dentro da matriz defibras de colagénio, que são cerca de 2 uM e 100 nm de diâmetro, respectivamente. Os fun1 manchadas de imagens do scanner microarray mostrar o fermento e as formas de hifas, que são característicos de biofilmes de fungos. As imagens 2D e 3D-confocal de fluorescência de fun1 coradas biofilmes pode ser visto como tendo heterogeneidade espacial, com as regiões de células metabolicamente activas intercaladas no interior da matriz extracelular (composto de colagénio como o material de encapsulação e muito provavelmente também de material exopolymeric produzido por células do biofilme), que não é coradas pelo corante metabólicas. A espessura do biofilme foi estimada em cerca de 50 uM. O BChip Ca foi utilizada para estimar a susceptibilidade antifúngica de dois fármacos, fluconazol e anfotericina B, e os resultados são mostrados na Figura 3. Consistente com relatórios publicados sobre ensaios padrão da indústria placa de 96 poços, os biofilmes são resistentes contra o fluconazol 10 e 50 do IC calculado para<html> anfotericina B é de 0,3 ug / mL 11. <img alt="A Figura 1" src="/files/ftp_upload/3845/3845fig1.jpg" /> Figura Fluxograma 1. Para a fabricação de Ca BChip. <img alt="A Figura 2" src="/files/ftp_upload/3845/3845fig2.jpg" /> Figura 2. A imagem do alto thoughput Ca BChip, impresso usando um microarrayer robótica e contendo 768 pontos em PS-MA-revestidos slides. Cada ponto é hemisférica 50 nL, em volume, cerca de 700 um de diâmetro, e com uma separação de 1,2 mm entre pontos. Também é mostrado (sentido horário) são microscopia de luz, scanner microarray, microscopia 2D e 3D-fluorescência, e digitalização de imagens de microscopia eletrônica dos biofilmes individuais na BChip Ca. A figura SEM com ampliação elevada de 25.000 x mostra um filamento de hifas de largura de 2 uM. Figura 3 &quot;src =&quot; files/ftp_upload/3845/3845fig3.jpg / &quot;/&gt; Figura 3. Resultados dos testes de susceptibilidade antifúngica e determinação de valores de IC50 para (A) fluconazol e (B) a anfotericina B, utilizando Ca BChip. As imagens de microscopia de fluorescência de anfotericina B tratadas com biofilmes são mostrados em (C). Os resultados são a média ± erro padrão para significar dois chips separados contendo 10 réplicas para cada condição.

<ol>
	<li>Edmond, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nosocomial+bloodstream+infections+in+United+States+hospitals:+a+three-year+analysis.">Nosocomial bloodstream infections in United States hospitals: a three-year analysis.</a> Clin. Infect. Dis. 29, 239-244 (1999).</li><li>Ramage, G., Bachmann, S., Patterson, T. F., Wickes, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+multidrug+efflux+pumps+in+relation+to+fluconazole+resistance+in+Candida+albicans+biofilms.">Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms.</a> J. Antimicrob. Chemother. 49, 973-980 (2002).</li><li>Srinivasan, A., Uppuluri, P., Lopez-Ribot, J., Ramasubramanian, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+High-Throughput+Candida+albicans+Biofilm+Chip.">Development of a High-Throughput Candida albicans Biofilm Chip.</a> PLoS ONE. 6, 19036-19036 (2011).</li><li>Ramage, G., Vandewalle, K., Wickes, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+biofilm+formation+by+Candida+albicans.">Characteristics of biofilm formation by Candida albicans.</a> Rev. Iberoam. Micol. 18, 163-170 (2001).</li><li>Pierce, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+reproducible+96-well+plate-based+method+for+the+formation+of+fungal+biofilms+and+its+application+to+antifungal+susceptibility+testing.">A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing.</a> Nat. Protoc. 3, 1494-1500 (2008).</li><li>Lee, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cellular+microarray+for+high-throughput+toxicology+assays.">Three-dimensional cellular microarray for high-throughput toxicology assays.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 59-63 (2008).</li><li>Meyerhofer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+resist+films+produced+by+spinning.">Characteristics of resist films produced by spinning.</a> Journal of Applied Physics. 49, (1978).</li><li>Ramage, G., Vande Walle, K., Wickes, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+method+for+in+vitro+antifungal+susceptibility+testing+of+Candida+albicans+biofilms.">Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms.</a> Antimicrob. Agents Chemother. 45, 2475-2479 (2001).</li><li>Chandra, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+by+the+fungal+pathogen+Candida+albicans:+Development,+architecture,+and+drug+resistance.">Biofilm formation by the fungal pathogen Candida albicans: Development, architecture, and drug resistance.</a> Journal of Bacteriology. 183, 5385-5394 (2001).</li><li>Jabra-Rizk, M. A., Falkler, W. A., Meiller, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungal+biofilms+and+drug+resistance.">Fungal biofilms and drug resistance.</a> Emerg. Infect. Dis. 10, 14-19 (2004).</li><li>Tobudic, S., Lassnigg, A., Kratzer, C., Graninger, W., Presterl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+activity+of+amphotericin+B,+caspofungin+and+posaconazole+on+Candida+albicans+biofilms+in+intermediate+and+mature+development+phases.">Antifungal activity of amphotericin B, caspofungin and posaconazole on Candida albicans biofilms in intermediate and mature development phases.</a> Mycoses. 53, 208-214 (2010).</li></ol>

Jose L. Lopez-Ribot e Anand K. equidade Ramasubramanian própria MicrobeHTS Technologies, Inc., que está desenvolvendo agentes antifúngicos. MicrobeHTS Technologies, Inc. não forneceu qualquer apoio financeiro para estes estudos.

Nós desenvolvemos uma célula baseado microarray de alta densidade, Ca BChip, consistindo em volumes nanolitros de biofilmes de Candida albicans. O microarray foi impresso em substratos de vidro modificado, o que permitiu a fixação robusta de pontos de gel de colágeno, proporcionando hidrofobicidade necessário para um não-divulgação, gel hemisférica 3D. Um único Ca BChip pode substituir cerca de oito placas de 96 alvéolos, e vários chips podem ser impressos e processado, ao mesmo t...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> 3-aminopropiltrietoxissilano (APTES) </td><td> Sigma-Aldrich </td><td> 440140 </td></tr><tr><td> Poliestireno-Co-anidrido maleico (PS-MA) </td><td> Sigma-Aldrich </td><td> 426946 </td></tr><tr><td> Microscopia lâminas de vidro </td><td> Fisher Scientific </td><td> 12-549-3 </td></tr><tr><td> Rato Cauda colágeno tipo I </td><td> BD Biosciences </td><td> 354236 </td></tr><tr><td> Microarrayer Robotic </td><td> Omnigrid Micro </td><td> MICROSYS4000/4100A </td></tr><tr><td> Microarray Scanner </td><td> GenePix pessoais 4100A </td><td> GENEPIX4100A </td></tr><tr><td> Cassete de hibridação </td><td> ArrayIt Corporação </td><td> AHCXD </td></tr><tr><td> Fun1 [2-clororo-4-(2,3-di-hidro-3-metil-(benzo -1,3-tiazol-2-il)-metilideno)-1-fenil quinoliniumiodide] </td><td> Invitrogen Corp </td><td> F-7030 </td></tr><tr><td> Fluconazol </td><td> SICOR Pharmaceuticals, Inc. </td><td> J02AC01 </td></tr><tr><td> A anfotericina B </td><td> Sigma </td><td> A2411 </td></tr><tr><td> RPMI-1640 </td><td> Mediatech, Inc. </td><td> 50-020-PC </td></tr><tr><td> Cerâmica Dica orifício mM 190 </td><td> Digilab </td><td> 60020441-00 </td></tr><tr><td> GraphPad Prism Software </td><td> GraphPad Software, Inc. </td><td></td></tr><tr><td> GenePix Pro V4.1 </td><td> Molecular Devices </td><td></td></tr></tbody></table>

candida albicans biofilm chip (cabchip) for high-throughput antifungal drug screening

Candida albicans continua a ser o principal agente etiológico da candidíase, que atualmente representa a infecção da corrente sangüínea quarta nosocomial mais comum em hospitais dos Estados Unidos 1. Estas infecções oportunistas são uma ameaça crescente para um número crescente de indivíduos comprometidos, e levar as taxas de mortalidade inaceitavelmente elevados. Isto é em parte devido ao arsenal limitado de drogas antifúngicas, mas também para o aparecimento de resistência contra os agentes mais utilizados antifúngicos. Para complicar ainda mais o tratamento é o facto de que uma maioria das manifestações de candidíase estão associados com a formação de biofilmes, e as células dentro destas biofilmes mostrar aumento dos níveis de resistência à maioria clinicamente utilizados agentes antifúngicos 2. Descrevemos aqui o desenvolvimento de uma micromatriz de alta densidade, que consiste de C. albicans nano-biofilmes, o que temos chamado Ca BChip 3. Resumidamente, um microarrayer robótico é utilizado tO impressão células de levedura de C. albicans sobre um substrato sólido. Durante a impressão, as células de levedura são colocadas em uma matriz tridimensional usando um volume tão baixo como 50 nL e imobilizada sobre um substrato de vidro com um revestimento adequado. Após a impressão inicial, as lâminas são incubadas a 37 ° C durante 24 horas para permitir a formação do biofilme. Durante este período, as manchas crescem em plenamente desenvolvidos &quot;nano-biofilmes&quot; que exibem características típicas estruturais e fenotípicos associados C. maduro albicans biofilmes (ie complexidade morfológica, três arquitetura dimensional e resistência às drogas) 4. Em geral, a BChip Ca é composto de ~ 750 biofilmes equivalentes e espacialmente distinto; com a vantagem adicional de que múltiplos chips podem ser impressos e processados ​​simultaneamente. A viabilidade celular é estimada pela medição da intensidade de fluorescência de fun1 metabólica mancha usando um scanner microarray. Este chip de fungos é ideal para usi na triagem de alto rendimento verdadeiro para a descoberta de drogas anti-fúngico. Em comparação com as normas em vigor (isto é, a de 96 poços modelo placa de microtitulação de biofilme formação 5), as principais vantagens do chip biofilme fúngica são automação, a miniaturização, a poupança em quantidade e custo dos reagentes e análises em tempo, bem como a eliminação do trabalho passos intensivos. Acreditamos que tais chips irá acelerar significativamente o processo de descoberta de drogas antifúngicas.

Watch this Scientific Journal Video about Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening at JoVE.com

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

Nós desenvolvemos uma plataforma de microarray de alta densidade que consiste em 3D nano-biofilmes de<em&gt; C. albicans</em&gt; Chamada<em&gt; Ca</em&gt; BChip. O perfil de susceptibilidade de drogas testadas em um<em&gt; Ca</em&gt; BChip é comparável com o modelo de placa convencional de 96 poços, sugerindo que o chip fúngica é idealmente adequado para o rastreio de alto rendimento real de drogas antifúngicas.

Candida albicans biofilme Chip (Ca BChip) para seleção de alto rendimento antifúngico

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

candida-albicans-biofilm-chip-cabchip-for-high-throughput-antifungal

Research

JoVE Journal

Bioengineering

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

13.6K visualizações.  University of Texas at San Antonio. Nós desenvolvemos uma plataforma de microarray de alta densidade que consiste em 3D nano-biofilmes de C. albicans Chamada Ca BChip. O perfil de susceptibilidade de drogas testadas em um Ca BChip é comparável com o modelo de placa convencional de 96 poços, sugerindo que o chip fúngica é idealmente adequado para o rastreio de alto rendimento real de drogas antifúngicas.

Vídeo: Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

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

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Microarrays de polímeros para Discovery High Throughput de Biomateriais

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

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 ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Usando Tomoauto: Um Protocolo de métodos de alta capacidade automatizada Cryo-Electron Tomografia

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Uma plataforma microfluídica para a isolação de alta taxa de transferência de célula única e Cultura

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

High-throughput identificação de bactérias Polymers Repelente para dispositivos médicos

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Automated Análise contração do Coração Humano engenharia de tecidos para Screening Cardiac Drug Safety

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

Microtissues 3D para terapia regenerativa injetável e triagem de drogas de alto rendimento

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Uso do elevado-Throughput Microbioreactor sistema automatizado para produção de IgG1 modelo em células CHO

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

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 ...