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

We willen graag G. O Toole bedanken voor de begeleiding en suggesties bij de ontwikkeling van deze modellen. Dit werk werd ondersteund door de Cystic Fibrosis Foundation (ANDERS06F0 naar GGA, STANTO07RO en STANTO08GA aan BAS), de National Institutes of Health (T32A107363 aan GGA en R01-HL074175 naar BAS), en het National Center for Research Resources Centers for Biomedical Research Excellence (Cobre P20-RR018787 aan BAS).

1. Static Co-cultuur Biofilm Model <ol><li> De Static Co-cultuur Biofilm model maakt gebruik van een CFBE41o-cellen (CFBE cellen), die zijn vereeuwigd cellen oorspronkelijk ontwikkeld vanuit een individu met CF homozygoot voor de ΔF508-CFTR mutatie 2,3,4. CFBE cellen moet worden uitgezet met een concentratie van 10 6 cellen / putje in een 6-well weefselkweek plaat of 2 X 10 5 in een 24-well plaat in weefselkweek minimaal essentieel medium (MEM) aangevuld met 10% foetaal run...

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	<li>Anderson, G. G., Moreau-Marquis, S., Stanton, B. A., 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=In+vitro+analysis+of+tobramycin-treated+Pseudomonas+aeruginosa+biofilms+on+cystic+fibrosis-derived+airway+epithelial+cells.">In vitro analysis of tobramycin-treated Pseudomonas aeruginosa biofilms on cystic fibrosis-derived airway epithelial cells.</a> Infect Immun. 76, 1423-1433 (2008).</li><li>Bruscia, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+CF+cell+lines+corrected+at+DeltaF508-CFTR+locus+by+SFHR-mediated+targeting.">Isolation of CF cell lines corrected at DeltaF508-CFTR locus by SFHR-mediated targeting.</a> Gene Ther. 9, 683-685 (2002).</li><li>Cozens, A. 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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Biofilms zijn gemeenschappen van bacteriën die vorm in reactie op prikkels uit de omgeving. Deze milieu-signalen leiden tot wereldwijde wijzigingen in de regelgeving binnen elke bacterie, wat resulteert in binding aan een oppervlak, aggregatie, de productie van exopolysacchariden, en andere fenotypes zoals verhoogde resistentie tegen antibiotica 10. In de afgelopen paar decennia, is er veel bewijs ondersteunde de hypothese dat biofilms een grote rol spelen in de pathogenese van chronische infecties. Zo is he...

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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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>
<tr>
<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>
<td>&nbsp;</td>
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<td>MEM without phenol red</td>
<td>&nbsp;</td>
<td>Mediatech, Manassass, VA</td>
<td>Mediatech, Manassass, VA</td>
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co-culture models of pseudomonas aeruginosa biofilms grown on live human airway cells

Bacteriële biofilms zijn geassocieerd met een aantal verschillende ziekten bij de mens, maar biofilm ontwikkeling is in het algemeen onderzocht op niet-levende oppervlakken. In dit artikel beschrijven we protocollen voor het vormen van Pseudomonas aeruginosa biofilms op menselijke epitheelcellen (CFBE cellen) in kweek. In de eerste methode (genoemd de Static Co-cultuur Biofilm Model), P. aeruginosa is geïncubeerd met CFBE cellen gekweekt als samenvloeiende monolagen op standaard weefselkweek platen. Hoewel de bacterie is zeer giftig voor de epitheelcellen, de toevoeging van arginine vertraagt ​​de vernietiging van de monolaag lang genoeg om biofilms te vormen op de CFBE cellen. De tweede methode (aangeduid als de Flow Cell Co-cultuur Biofilm Model), omvat de aanpassing van een biofilm flow cel apparatuur, die vaak wordt gebruikt in de biofilm onderzoek, om een ​​glazen dekglaasje aan het ondersteunen van een confluente monolaag van cellen CFBE tegemoet te komen. Deze monolaag wordt geënt met P. aeruginosa en een peristaltische pomp stroomt dan vers medium over de cellen. In beide systemen, bacteriën biofilms vormen binnen 6-8 uur na inoculatie. Visualisatie van de biofilm wordt versterkt door het gebruik van P. aeruginosa stammen constitutief uiten van groen fluorescerend eiwit (GFP). De statische en Flow Cell Co-cultuur Biofilm assays zijn modelsystemen voor de vroege P. aeruginosa infectie van de Cystic Fibrosis (CF) long-, en deze technieken laat verschillende aspecten van P. aeruginosa biofilmvorming en virulentie te bestuderen, met inbegrip van biofilm cytotoxiciteit, meting van de biofilm CFU en kleuring en visualiseren van de biofilm.

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

Dit artikel beschrijft verschillende methoden van groeiende<em&gt; Pseudomonas aeruginosa</em&gt; Biofilms op gekweekte menselijke epitheelcellen. Deze protocollen kunnen worden aangepast aan verschillende aspecten van biofilmvorming, waaronder visualisatie van de biofilm studie, kleuring van de biofilm, het meten van de kolonievormende eenheden (CFU) van de biofilm, en het bestuderen van biofilm cytotoxiciteit.

Co-cultuur modellen van Pseudomonas aeruginosa Biofilms Grown op Live menselijke luchtwegen cellen

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

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