PMC:7025480 / 42739-45713 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7025480","sourcedb":"PMC","sourceid":"7025480","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7025480","text":"P. aeruginosa Strains and Virus Infections\nVarious P. aeruginosa strains (Table 1) were cultured in lysogeny broth (LB) broth overnight at 37°C, shaking at 200 rpm. The culture was then diluted 1:50 in RPMI-1640 medium and grown for 5 days at 37°C, shaking at 200 rpm, in order to activate quorum-sensing and virulence factor secretion. On day 5, the medium was centrifuged for 10 min at 4,200 rcf and passed through a 0.2 μm filter. This medium was used as conditioned medium (CM). For each experiment at least two different CM were used. A RPMI-1640 control was cultured and filtered alongside the cultured P. aeruginosa strains. 100,000 BEAS2B or primary cells were seeded in 24-well-plates in BEAS2B medium. The following day, the cell medium was changed to infection medium, which was RPMI-1640 supplemented with 2% FCS, 1% P/S, 25 mM HEPES, and 0.075% NaHCO3. Twenty-four hours later, BEAS2B were pretreated with CM (20% v/v) for 1 h at 37°C, 5% CO2. Cell medium was then removed, and respiratory syncytial virus (RSV) or rhinovirus (RV1B) at a multiplicity of infection of 1 (MOI 1) (stocks prepared in house) in combination with fresh CM was added to BEAS2B cells. After 1 h at room temperature, shaking at 30 rpm, medium was removed, cells washed twice with warm 1X PBS, and infection medium added to the cells. Fresh CM was once again added to the cells as indicated above. Cell culture supernatant was collected at the indicated times and cells were lysed in RNA lysis buffer.\nTable 1 Strain descriptions.\nNo. Strain Description and characteristics References\n1 PAO1 Pseudomonas aeruginosa wild type DSMZ 22644\n2 Boston Pseudomonas aeruginosa wild type ATCC 27853\n3 PA14 Pseudomonas aeruginosa wild type DSMZ 19882\n4 PA7 Pseudomonas aeruginosa wild type DSMZ 24068\n5 CF1.1 CF patient isolate (6, 40)\n6 CF1.2 CF patient isolate, clonally related to 5 (6, 40)\n7 CF2.1 CF patient isolate (6, 40)\n8 CF2.2 CF patient isolate, clonally related to 7 (6, 40)\n9 CF3.1 CF patient isolate (6, 40)\n10 CF3.2 CF patient isolate, clonally related to 9 (6, 40)\n11 CF4.1 CF patient isolate (6, 40)\n12 CF4.2 CF patient isolate, clonally related to 11 (6, 40)\n13 CF1 CF patient isolate (6, 40)\n14 CF1ΔlasR CF1 lasR deletion mutant (6, 40)\n15 CF2 CF patient isolate (6, 40)\n16 CF2ΔlasR CF2 lasR deletion mutant (6, 40)\n17 CF3 CF patient isolate (6, 40)\n18 CF3ΔlasR CF3 lasR deletion mutant (6, 40)\n19 CF4 CF patient isolate (6, 40)\n20 CF4ΔlasR CF4 lasR deletion mutant (6, 40)\n21 CF5 CF patient isolate (6, 40)\n22 CF5ΔlasR CF5 lasR deletion mutant (6, 40)\n23 PA14 Pseudomonas aeruginosa wild type parental strain of 24, 25, 26, 27 (43, 67)\n24 PA14ΔAprA PA14 transposon insertion mutant, ID23768 (43, 68)\n25 PA14ΔLasA PA14 transposon insertion mutant, ID35267 (43, 68)\n26 PA14ΔLasB PA14 transposon insertion mutant, ID31938 (43, 68)\n27 PA14ΔPrpL PA14 transposon insertion mutant, ID37740 (43, 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