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

Vi vill tacka G. O Toole för vägledning och förslag för att utveckla dessa modeller. Detta arbete stöddes av cystisk fibros stiftelsen (ANDERS06F0 till GGA, STANTO07RO och STANTO08GA till BAS), National Institutes of Health (T32A107363 till GGA och R01-HL074175 till BAS) och National Center for Forskning Centers for Biomedical Research Excellence (Cobre P20-RR018787 till BAS).

1. Statisk Co-kulturen Biofilm Modell <ol><li> Den Static Co-kultur Biofilm modell 1 använder CFBE41o-celler (CFBE celler), som är förevigade celler ursprungligen utvecklats från en individ med CF homozygota för ΔF508-CFTR mutationer 2,3,4. CFBE celler bör seedas i en koncentration av 10 6 celler / brunn i en 6-och vävnadsodling platta eller 2 x 10 5 i 24-och vävnadsodling plattan i minimala viktigt medium (MEM) kompletteras med 10% fetalt bovint serum, 2mm L-glutam...

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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. 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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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Biofilmer är samhällen av bakterier som bildas som svar på stimuli från omgivningen. Dessa miljö-signaler leder till globala förändringar av regelverket inom varje bakterie, vilket resulterar i att binda till en yta, sammanställning, produktion av exopolysaccharides, och andra fenotyper som ökad antibiotikaresistens 10. Under de senaste decennierna har mycket bevis stöder hypotesen att biofilmer spelar en stor roll i patogenesen av kroniska infektioner. Till exempel är det väl accepterat att P...

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<td>FCS2 (Focht Live-Cell) chamber</td>
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<td>40 mm glass coverslips</td>
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<td>Bioptechs, Butler, PA</td>
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<td>MEM without phenol red</td>
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co-culture models of pseudomonas aeruginosa biofilms grown on live human airway cells

Bakteriella biofilmer har förknippats med en rad olika mänskliga sjukdomar, men biofilm utvecklingen i allmänhet har studerats på icke-levande ytor. I denna skrift beskriver vi protokoll för att bilda Pseudomonas aeruginosa biofilmer på människors luftvägar epitelceller (CFBE celler) odlas i kultur. I den första metoden (sk Static Co-kultur Biofilm Model), P. aeruginosa är inkuberas med CFBE celler odlas som konfluenta monolager på vanliga tallrikar vävnadsodling. Även om bakterien är mycket giftigt för epitelceller, tillägg av arginin förseningar förstörelsen av cellslager tillräckligt länge för biofilmer till form på CFBE celler. Den andra metoden (sk Flow Cell Co-kultur Biofilm Model), innebär anpassning av en biofilm flödescell apparater, som ofta används i biofilmen forskning, för att rymma ett glas täckglas stödja en konfluenta monolager av CFBE celler. Detta cellslager är inokuleras med P. aeruginosa och en Slangpumpar rinner sedan färska medelstora över de celler. I båda systemen, bakteriella biofilmer form inom 6-8 timmar efter ympningen. Visualisering av biofilmen förstärks genom användning av P. aeruginosa-stammar som uttrycker konstitutivt grönt fluorescerande protein (GFP). De statiska och Flow Cell Co-kulturen Biofilm analyser modellsystem för tidig P. aeruginosa infektion i cystisk fibros (CF) lunga, och dessa tekniker möjliggör olika aspekter av P. aeruginosa biofilm bildning och virulens som skall studeras, inklusive biofilmen cytotoxicitet, mätning av biofilm CFU och färgning och visualisera biofilmen.

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

Detta dokument beskriver olika metoder att växa<em&gt; Pseudomonas aeruginosa</em&gt; Biofilmer på odlade humana luftvägar epitelceller. Dessa protokoll kan anpassas för att studera olika aspekter av biofilm bildning, inklusive visualisering av biofilm, färgning av biofilm, mäta kolonibildande enheter (CFU) av biofilm, och studera biofilm cytotoxicitet.

Co-kultur modeller av Pseudomonas aeruginosa Biofilmer odlas på levande mänskliga Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

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

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