Name: co-culture models of pseudomonas aeruginosa biofilms grown on live human airway cells
Uploaded: 2010-10-06T17:00:00
Description: Watch this Scientific Journal Video about Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells at JoVE.com

Gostaríamos de agradecer a G. O Toole para orientação e sugestões no desenvolvimento destes modelos. Este trabalho foi financiado pela Cystic Fibrosis Foundation (ANDERS06F0 ao GGA, STANTO07RO e STANTO08GA a BAS), os Institutos Nacionais de Saúde (T32A107363 para GGA e R01-HL074175 a BAS) e do Centro Nacional de Centros de Pesquisa de Recursos para a Excelência Investigação Biomédica (COBRE P20-RR018787 a BAS).

1. Modelo Biofilm estática co-cultura <ol><li> O modelo de biofilme estática cultura Co-1 usa CFBE41o células (células CFBE), que são imortalizadas células originalmente desenvolvido a partir de um indivíduo com homozigotos CF para a mutação DF508-CFTR 2,3,4. Células CFBE deve ser semeado a uma concentração de 10 6 células / poço em uma placa de cultura de 6 bem tecido ou 2 X 10 5 em uma placa de cultura de 24 poços de tecidos em meio essencial mínimo (MEM) s...

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFTR+expression+and+chloride+secretion+in+polarized+immortal+human+bronchial+epithelial+cells.">CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells.</a> Am J Respir Cell Mol Biol. 10, 38-47 (1994).</li><li>Hentchel-Franks, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+airway+cl-+secretion+in+human+subjects+by+adenosine.">Activation of airway cl- secretion in human subjects by adenosine.</a> Am J Respir Cell Mol Biol. 31, 140-146 (2004).</li><li>Moreau-Marquis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DeltaF508-CFTR+mutation+results+in+increased+biofilm+formation+by+Pseudomonas+aeruginosa+by+increasing+iron+availability.">The DeltaF508-CFTR mutation results in increased biofilm formation by Pseudomonas aeruginosa by increasing iron availability.</a> Am J Physiol Lung Cell Mol Physiol. 295, 25-37 (2008).</li><li>Kuchma, S. L., Connolly, J. P., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-component+regulatory+system+regulates+biofilm+maturation+and+type+III+secretion+in+Pseudomonas+aeruginosa.">A three-component regulatory system regulates biofilm maturation and type III secretion in Pseudomonas aeruginosa.</a> J Bacteriol. 187, (2005).</li><li>Heydorn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+reproducibility+in+flow-chamber+biofilms.">Experimental reproducibility in flow-chamber biofilms.</a> Microbiology. 146 (Pt 10), 2409-2415 (2000).</li><li>Heydorn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT.">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146 (Pt 10), 2395-2407 (2000).</li><li>Anderson, G. 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Biofilmes são comunidades de bactérias que se formam em resposta a estímulos ambientais. Estes sinais ambientais globais levar a mudanças regulatórias dentro de cada bactéria, resultando na ligação a uma superfície, de agregação, a produção de exopolissacarídeos, e outros fenótipos, tais como aumento da resistência aos antibióticos 10. Ao longo das duas últimas décadas, muitas provas apoiou a hipótese de que biofilmes desempenham um grande papel na patogênese das infecções crônicas. Po...

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<td>FCS2 (Focht Live-Cell) chamber</td>
<td>&nbsp;</td>
<td>Bioptechs, Butler, PA</td>
<td>060319131616</td>
<td>&nbsp;</td>
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<td>FCS2 chamber controller</td>
<td>&nbsp;</td>
<td>Bioptechs, Butler, PA</td>
<td>060319-2-0303</td>
<td>&nbsp;</td>
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<td>40 mm glass coverslips</td>
<td>&nbsp;</td>
<td>Bioptechs, Butler, PA</td>
<td>PH 40-1313-0319</td>
<td>&nbsp;</td>
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<td>MEM</td>
<td>&nbsp;</td>
<td>Mediatech, Manassass, VA</td>
<td>#10-010-CV</td>
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<td>MEM without phenol red</td>
<td>&nbsp;</td>
<td>Mediatech, Manassass, VA</td>
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co-culture models of pseudomonas aeruginosa biofilms grown on live human airway cells

Biofilme bacteriano têm sido associados com um número de diferentes doenças humanas, mas o desenvolvimento de biofilme geralmente tem sido estudada em superfícies não-vivos. Neste artigo, descrevemos os protocolos para formar biofilmes de Pseudomonas aeruginosa nas células epiteliais das vias aéreas humanas (células CFBE) cultivadas em cultura. No primeiro método (chamado de modelo de biofilme estática Co-cultura), P. aeruginosa é incubada com células CFBE crescido como monocamadas confluentes em placas de cultura de tecido normal. Embora a bactéria é muito tóxico para as células epiteliais, a adição de atrasos arginina a destruição da monocamada tempo suficiente para formar biofilmes sobre as células CFBE. O segundo método (chamado de fluxo de células Modelo Biofilm Co-cultura), envolve a adaptação de um aparelho celular biofilme fluxo, que é frequentemente usado em biofilme de pesquisa, para acomodar uma lamela de vidro apoiar uma monocamada confluente de células CFBE. Este monocamada é inoculado com P. aeruginosa e uma bomba peristáltica, então, flui meio fresco através das células. Em ambos os sistemas, biofilme bacteriano forma dentro de 6-8 horas após a inoculação. Visualização do biofilme é reforçada pelo uso de P. cepas aeruginosa constitutivamente expressando a proteína verde fluorescente (GFP). A estática e fluxo de células ensaios Co-cultura de biofilme são sistemas modelo para P. início aeruginosa infecção do pulmão da fibrose cística (CF), e estas técnicas permitem diferentes aspectos da P. formação de biofilme e virulência aeruginosa a ser estudado, incluindo a citotoxicidade biofilme, a medição do biofilme UFC, e de coloração e visualização do biofilme.

Watch this Scientific Journal Video about Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells at JoVE.com

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Este artigo descreve métodos diferentes de crescimento<em&gt; Pseudomonas aeruginosa</em&gt; Biofilmes em cultura de células epiteliais das vias aéreas humanas. Estes protocolos podem ser adaptados para estudar diferentes aspectos da formação de biofilme, incluindo a visualização do biofilme, a coloração do biofilme, medindo a unidades formadoras de colônias (CFU) do biofilme e estudar a citotoxicidade biofilme.

Co-cultura de Modelos Pseudomonas aeruginosa Biofilmes Grown no Live Células Airway Humanos

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