Agradecemos ao laboratório O&#39;Bryan a UIC para avaliação experimental crítica e discussão. Este trabalho foi financiado pela American Heart Association Grant 11SDG5230003 ao Centro GKC e Nacional para o avanço da ciência translacional - Centro de UIC para Ciências Clínicas e Translational Grant UL1TR000050.

1. Transfecção celular <ol><li> Células da placa um dia antes da transfecção, chapeamento menos células de imunofluorescência. <ol><li> Para imunofluorescência: em uma placa de 6 poços, casaco lamínulas de vidro (um por poço), com 0,02% de gelatina a 37 ° C durante pelo menos 4 horas, lave uma vez com o meio, e para cada poço, a placa 1 x 10 5 HEK 293T células em 2 ml de meio de crescimento completo [meio de Eagle modificado por Dulbecco (DMEM) mais 10% de FBS]. </li><li> Para a citometria de fluxo e análise da mancha Ocidental: placa de 1,2 x 10 5 células HEK 293T / poço em 2 ml de meio de crescimento completo na placa de 6 poços (0,3 x 10 5 células HEK 293T em 0,5 ml de meio de crescimento completo por poço em placas de 24 poços ). </li></ol></li><li> Prepare o ADN para transfecção de acordo com o protocolo do fabricante: Effectene (Qiagen) é utilizado para imunofluorescência. Trânsito LT1 (Mirus) é usado para análises de fluxo de blot e citometria ocidental. Quantidade de ADN utilizada é listado abaixo. <table border="1"><tbody><tr><td&gt; </td><td> Placa de 24 poços (ng) </td><td> Placa de 6 poços (ng) </td></tr><tr><td> PCP-vector </td><td> 10 </td><td> 50 </td></tr><tr><td> VN N-AKAP-Lbc </td><td> 200 </td><td> 1000 </td></tr><tr><td> VC N-PDE4D3-FL (full-length PDE4D3) </td><td> 2,5, 5, 10, 20, 40, 80 </td><td> 100 </td></tr><tr><td> VC-N-PDE4D3 UCR1 (PDE4D3 um fragmento N-terminal contém o domínio UCR1) </td><td></td><td> 100 </td></tr><tr><td> VC-N-PDE4D3 UCR2 + CAT (fragmento C-terminal contendo os domínios PDE4D3 UCR2 e catalítico) </td><td></td><td> 100 </td></tr></tbody></table> A transfecção utilizando Effectene (6 poços): <ol><li> Adicionar quantidade indicada de DNA de plasmídeo + 4 ul potenciador de 100 ul de tampão CE, misturar e incubar durante 5 min à temperatura ambiente. </li><li> Adicionar 5 uL Effectene, mistura, e incubar durante 10 min atemperatura ambiente. Adicionar gota a gota, para as células. </li></ol> Transfecção utilizando trânsito-LT1 (6-well/24-well): <ol><li> Adicionar quantidade indicada de DNA de plasmídeo a 250 μl/50 ul de Opti-MEM I meio livre de soro em um tubo estéril. </li><li> Adicionar trânsito LT1-reagente para a mistura de DNA diluído e gentilmente pipeta para misturar. (Transit-LT1 Reagente: relação DNA é de 3 ml de trânsito-LT1 reagente por 1 mg de DNA). </li><li> Incubar por 30 minutos em temperatura ambiente e adicionar gota a gota, para as células. </li></ol></li></ol> 2. Preparação de células para citometria de fluxo, Western Blot e Imunofluorescência <ol><li> 24 horas após a transfecção, verifique fluorescência sob microscópio de epifluorescência, antes de células colheita. </li><li> Preparação de células para análise por citometria de fluxo: <ol><li> Lavar as células com 0,5 ml / 2 ml de PBS gelado para placas 24-well/6-well. </li><li> Retire células usando 50 mL μl/250 0,05% de tripsina. </li><li> Neutraliza-se com 200 ul de tripsina / 1 ml de meio DMEM. </li><li> Collect 250 ul de células (tanto para placa de 24 poços e 6 poços) por centrifugação a 500 xg durante 5 min, utilizar as células restantes (1 mL) para análise de Western blot. </li><li> Ressuspender as células em 250 ul de tampão FACS [PBS + 0,5% (w / v) de BSA + 2 mM de EDTA], armazenar em gelo para análise de citometria de fluxo. As células também podem ser fixados em paraformaldeído a 1% (em PBS), se a análise de citometria de fluxo não será imediata. </li></ol></li><li> Preparação de células para análise de Western blot, realizado em paralelo com a preparação de células para análise de citometria de fluxo. <ol><li> Lavar as células com 2 ml de 1x PBS gelado, lise de células por adição de 100 ul de tampão de lise celular [tampão 10 mM de fosfato de sódio, pH 6,95, 150 mM NaCl, 5 mM de EDTA, 5 mM de EGTA, 1% de Triton X-100] . </li><li> Recolhe ligado celular por centrifugação durante 10 min a 21.000 xg, a 4 ° C. </li><li> Quantificar a concentração de proteína pelo ensaio de proteína Bradford, coloque a mesma quantidade de proteína por pista para SDS-PAGE, e transferir para nitrocelulose por immunoblotting. </li></ol&gt; </li><li> Preparação de células para imunofluorescência. <ol><li> Lavar as células 3x com 2 ml de PBS, as células fixar em 1 ml de 3,7% de paraformaldeído (em PBS) durante 10 min à temperatura ambiente, em seguida, lavar 3x com 2 ml de PBS durante 5 min. </li><li> Monte cobrir deslizamento em uma lâmina de vidro, utilizando-se de meio de montagem. Selar as bordas lamela usando unha polonês, se necessário. </li><li> Armazenar as lâminas a 4 ° C antes da análise. </li></ol></li></ol> 3. Citometria de fluxo e microscopia de imunofluorescência <ol><li> A citometria de fluxo é realizada utilizando um Beckman Coulter Ciano ADP, equipado com 488 nm, 405 nm, 642 nm e lasers de estado sólido. Software Summit é utilizado para aquisição e análise. <ol><li> Calibrar citômetro de fluxo, utilizando contas padrão. </li><li> Gráfico de dispersão para a frente (FSC) / para o lado de dispersão (SSC) em uma escala linear para gating população de células vivas. </li><li> Plot largura de pulso FSC / tensão para gating população de células vivas não agregada. </li><li> Plot count/FL6 (PCP fluorescence, 405 nm) numa escala logarítmica para gating células não agregadas vivos PCP-positivos. </li><li> Plot count/FL1 (YFP fluorescência, 488 nm) em uma escala logarítmica para a intensidade BiFC. </li><li> Executar amostras YFP-CFP-dupla negativa, ajuste FSC, SSC, FL1 e FL6 foto tubo multiplicador (PMT) de tensão para definir o limite para cada sinal. </li><li> Executar único positivo (ambos YFP + e PCP +) amostras para futura compensação off-line </li><li> Adquirir pelo menos 10.000 eventos fechados (células não agregados ao vivo PCP +) para análise. </li></ol></li><li> Microscopia de imunofluorescência é realizada utilizando uma Zeiss LSM 510 microscópio confocal Nota:. Que é importante para manter todas as configurações (como o zoom, pinhole, ganho de detector, amplificador offset, tamanho do quadro, velocidade de digitalização, digitalização média e potência do laser) consistente entre amostras para comparação adequada de todos os dados. </li></ol> 4. Análise de Dados (realizada utilizando Summit Software) <ol><li> Configure o protocolo de análise semelhante à acquisitíon com gating adequada: Gate 1 em FSC / SSC para as células vivas. Gate 2 em FSC / largura de pulso do Portão 1 para as células vivas não agregados. Portão 3 em contagem / CFP do Portão 3 para o PCP não agregada + células vivas. </li><li> Compensar YFP e CFP sinal usando YFP + e + PCP células positivas individuais. </li><li> Re-determinam linhas de corte para as células CFP / YFP-positivos. </li><li> Exportação YFP intensidade média de fluorescência (MFI) de PCP + células para o Excel para dados de plotagem. </li><li> Normalizar YFP a níveis de IFM-AKAP Lbc, PDE4D3 expressão, e carregamento de controlo α-tubulina, tal como determinado por Western blot. </li></ol>

<ol>
	<li>Collura, V., Boissy, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+protein-protein+complexes+to+interactomics.">From protein-protein complexes to interactomics.</a> Subcell Biochem. 43, 135-183 (2007).</li><li>Stumpf, M. P., 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=Estimating+the+size+of+the+human+interactome.">Estimating the size of the human interactome.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 6959-6964 (2008).</li><li>Shekhawat, S. S., Ghosh, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split-protein+systems:+beyond+binary+protein-protein+interactions.">Split-protein systems: beyond binary protein-protein interactions.</a> Curr. Opin. Chem. Biol. 15, 789-797 (2011).</li><li>Dwane, S., Kiely, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+used+to+study+how+protein+complexes+are+assembled+in+signaling+cascades.">Tools used to study how protein complexes are assembled in signaling cascades.</a> Bioeng. Bugs. 2, 247-259 (2011).</li><li>Piehler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+methodologies+for+measuring+protein+interactions+in+vivo+and+in+vitro.">New methodologies for measuring protein interactions in vivo and in vitro.</a> Curr. Opin. Struct. Biol. 15, 4-14 (2005).</li><li>Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+implementation+of+bimolecular+fluorescence+complementation+(BiFC)+assays+for+the+visualization+of+protein+interactions+in+living+cells.">Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.</a> Nat. Protoc. 1, 1278-1286 (2006).</li><li>Shyu, Y. J., Suarez, C. D., Hu, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+ternary+complexes+in+living+cells+by+using+a+BiFC-based+FRET+assay.">Visualization of ternary complexes in living cells by using a BiFC-based FRET assay.</a> Nat. Protoc. 3, 1693-1702 (2008).</li><li>Wong, K. A., O'Bryan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+fluorescence+complementation.">Bimolecular fluorescence complementation.</a> J. Vis. Exp. (50), e2643 (2011).</li><li>Sugarbaker, E. V., Thornthwaite, J. T., Temple, W. T., Ketcham, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+general+principles+and+applications+to+selected+studies+in+tumor+biology.">Flow cytometry: general principles and applications to selected studies in tumor biology.</a> Int. Adv. Surg. Oncol. 2, 125-153 (1979).</li><li>Koshy, S., Alizadeh, P., Timchenko, L. T., Beeton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+GLUT4+translocation+to+the+plasma+membrane+by+flow+cytometry.">Quantitative measurement of GLUT4 translocation to the plasma membrane by flow cytometry.</a> J. Vis. Exp. (45), e2429 (2010).</li><li>Smith, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+confocal+microscopy.">Basic confocal microscopy.</a> Curr. Protoc. Mol. Biol. Chapter 14 (Unit 14), 11 (2008).</li><li>Westwick, J. K., Lamerdin, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+drug+discovery+with+contextual+assays+and+cellular+systems+analysis.">Improving drug discovery with contextual assays and cellular systems analysis.</a> Methods Mol. Biol. 756, 61-73 (2011).</li><li>Kilpatrick, L. E., Holliday, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+pharmacology+of+G+protein-coupled+receptor+signaling+complexes+using+bimolecular+fluorescence+complementation.">Dissecting the pharmacology of G protein-coupled receptor signaling complexes using bimolecular fluorescence complementation.</a> Methods Mol. Biol. 897, 109-138 (2012).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

BiFC é um método simples e sensível para estudar a interacção proteína-proteína. Este método não pode ser utilizado para identificar novas interacções proteína-proteína, no entanto, é especialmente conveniente para confirmar a interacção proteína-proteína no interior das células e para estudar as propriedades funcionais, tais como a localização subcelular de complexos de proteínas, mapeamento de sítios de interacção proteína-proteína, e para o rastreio de péptidos / moléculas pequenas que po...

650.000 de interacções proteína-proteína são estimadas a existir no interactoma humano, desempenham papéis críticos para manter as funções normais da célula 1,2. Além de co-imunoprecipitação (co-PI), o padrão de ouro para estudar a interacção proteína-proteína a partir de lisados ​​de células, vários ensaios de complementação fragmento de proteína (APC) foram desenvolvidos para melhorar a sensibilidade na detecção de interacção proteína-proteína no interior das células 3. As técnicas incluem a transferência de energia por ressonância de Förster (FRET), bioluminescência Resonance Energy Transfer (BRET) e Complementação de fluorescência Bimolecular (BiFC) 4,5. BiFC é baseada na associação facilitada de dois fragmentos de uma proteína fluorescente (aqui usamos Vénus; uma variante YFP) que são, cada um fundido com um parceiro de proteína interagindo potencial (neste exemplo utilizamos AKAP-Lbc e PDE4D3). A interacção das duas proteínas de interesse dentro de células resulta em fluorescência funcional, que podem ser visualizadas por fmicroscopia luorescence usando tanto células vivas ou fixo 6,7,8. Comparado a outros PCAs, BiFC é sensível e menos tecnicamente desafiador, com potencial para estudar a localização celular em células vivas. Um grande inconveniente desta técnica é que, no entanto, uma vez formado, o complexo de proteína fluorescente não pode ser revertida. Portanto, não é um bom método para estudar a interacção proteína-proteína dinâmico. Neste trabalho, usamos BiFC para mapear os sítios de interação de PDE4D3 com AKAP-Lbc, monitorando as intensidades de fluorescência de Vênus. Em comparação com a análise tradicional utilizando a microscopia de fluorescência, o que é demorado e trabalhoso (se não for realizado utilizando maquinaria de alta taxa de transferência automatizada), citometria de fluxo, fornece uma análise quantitativa e simples dos milhares de células numa população heterogénea durante um curto período de tempo 9, 10. Aqui, através da realização de uma comparação lado-a-lado da análise de citometria de fluxo-BiFC tradicional e co-PI, que demonstram que os dois métodos proporcionam dados comparáveis, no entanto, a análise de fluxo citométrico BiFC é menos demorado e utiliza menos material, que pode ser mais útil quando as células de interesse são limitadas.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Dulbecco's modified Eagle's media (DMEM)</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin (pen/strep), 100x</td>
			<td>Invitrogen</td>
			<td>15070-063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-Flag antibody</td>
			<td>Sigma</td>
			<td>M2, F1804</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-HA antibody</td>
			<td>Covance</td>
			<td>16B12, MMS-101R</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&alpha;-tubulin</td>
			<td>Sigma</td>
			<td>DM1A, T9026</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Clontech</td>
			<td>632569</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell culture plates, 6-well tissue culture treated</td>
			<td>Thermo Fisher Scientific</td>
			<td>130184</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEK 293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Maxiprep plasmid purification kit, high speed</td>
			<td>Qiagen</td>
			<td>12663</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Phosphate-buffered saline (DPBS), sterile 1x</td>
			<td>Invitrogen</td>
			<td>14190-144</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.05% (w/v)</td>
			<td>Gibco</td>
			<td>25300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polystyrene round-bottom tubes for FACS staining</td>
			<td>BD Biosciences</td>
			<td>352052</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>S74337MF</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Prolong gold antifade reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P-36931</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superfrost plus Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fisherfinest premium cover glass</td>
			<td>Fisher Scientific</td>
			<td>12-548-5P</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>CO2 air-jacketed Incubator</td>
			<td>NuAIR DH autoflow</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Confocal microscope LSM510 META</td>
			<td>Carl Zeiss, Inc</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electrophoresis and transfer unit</td>
			<td>Biorad</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cyan ADP Flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

flow cytometric analysis of bimolecular fluorescence complementation: a high throughput quantitative method to study protein-protein interaction

Entre os métodos para estudar a interação proteína-proteína dentro das células, Complementação de fluorescência Bimolecular (BiFC) é relativamente simples e sensível. BiFC baseia-se na produção de fluorescência utilizando-se dois fragmentos não fluorescentes de uma proteína fluorescente (Vénus, uma variante de proteína fluorescente amarela, é aqui utilizada). Fragmentos de Vénus não fluorescente (VN e VC) são fundidos a duas proteínas que interagem (neste caso, AKAP-Lbc e PDE4D3), produzindo fluorescência devido à interacção VN-AKAP-LBC-VC-PDE4D3 e a formação de uma proteína fluorescente funcional no interior das células. BiFC fornece informações sobre a localização subcelular de complexos de proteínas e a intensidade das interacções de proteínas com base na intensidade de fluorescência. No entanto, a análise por microscopia de BiFC para quantificar a força da interacção proteína-proteína é demorado e subjectivos, devido à heterogeneidade na expressão da proteína e interacção. Acoplando citometria de fluxoA análise com a metodologia BiFC, o sinal BiFC interacção proteína-proteína fluorescente pode ser medida com precisão para uma grande quantidade de células num curto espaço de tempo. Aqui, nós demonstramos uma aplicação desta metodologia para mapear regiões PDE4D3 que são necessárias para a interação com AKAP-Lbc. Esta metodologia de elevado rendimento pode ser aplicado a factores de rastreio que regulam a interacção proteína-proteína.

AKAP-Lbc e PDE4D3 construções (Figura 1B) foram fundidas com fragmentos de VN e VC, respectivamente. Interacção dos AKAP-Lbc e PDE4D3 funcional resulta em YFP fluorescência (Figura 1A). Aqui usamos o método BiFC para mapear locais AKAP-LBC-vinculativos PDE4D3. Upstream uma região conservada (UCR1), a região a montante conservada 2 (UCR2) e região catalítica (CAT) são conservados entre as proteínas da família da PDE4, por conseguinte, o comprimento total (FL), e UCR1 UCR2 ma...

Watch this Scientific Journal Video about Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction at JoVE.com

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Citometria de fluxo e de Complementação de fluorescência Bimolecular fornece um método quantitativo de alto rendimento para estudar a interação proteína-proteína. Esta metodologia pode ser aplicada ao mapeamento de locais de ligação de proteína e para o rastreio de factores que regulam a interacção proteína-proteína.

Citometria de fluxo e de Complementação de fluorescência Bimolecular: A alta taxa de transferência método quantitativo para estudo de interação proteína-proteína

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

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18.3K visualizações.  University of Illinois at Chicago. Citometria de fluxo e de Complementação de fluorescência Bimolecular fornece um método quantitativo de alto rendimento para estudar a interação proteína-proteína. Esta metodologia pode ser aplicada ao mapeamento de locais de ligação de proteína e para o rastreio de factores que regulam a interacção proteína-proteína.

Vídeo: Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A Neuronal e astrocitária Ensaio Co-Cultura de Análise de Conteúdo de Alta de neurotoxicidade

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biologia

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

Um método de alto rendimento para estudar a morfologia global organela em S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Bimolecular de fluorescência de Complementação Assay (BiFC) para Interação Proteína-Proteína em células de cebola Utilizando o Gene Gun Helios

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

Complementação de fluorescência bimolecular

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

Detecção de interações proteína na planta através de um gateway de Complementação de fluorescência Compatível Bimolecular (BiFC) Sistema

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

A fase líquida ensaio de captura de afinidade usando pérolas magnéticas para estudar interação proteína-proteína: O Exemplo Poliovírus Nanobody-

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Análise Quantitativa de Autophagy usando microscopia de fluorescência avançada 3D

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Um Ensaio de Ligação de Alto Rendimento II do MHC para análise quantitativa de Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Interações proteína-proteína visualizado por Bimolecular Fluorescência de Complementação em tabaco Protoplastos e folhas

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

De alta capacidade Análise de Imagem de esferóides tumorais: uma aplicação de software de fácil utilização para medir o tamanho de esferóides automaticamente e com precisão

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...