Genequant pro spectrophotometer

RNA was extracted using a standard Trizol procedure (Life Technologies, UK, 15596-026). RNA samples were quantified by 260/280 nm absorption on a Gene Quant Pro spectrophotometer (Amersham Biosciences, UK) and analysed by gel electrophoresis. First-strand cDNA was synthesised from 5 μg of total RNA using random hexamer primers, following the manufacturer’s protocol (Cloned AMV First-strand cDNA Synthesis Kit, Life Technologies, 12328-040). Residual primers, nucleotides and enzymes were removed from the cDNA with a PCR purification kit (QIAquick PCR Purification Kit, QIAGEN, UK, 28106) following the manufacturer’s instructions.

The clinical NH57388A strain was provided by N. Hoffmann (University of Copenhagen). This strain possesses a mutation in mucA that results in hyperproduction of alginate (14 (link)). The mucoid YH5 strain and nonmucoid GRI-1 strain were obtained locally, from a patient with CF and a patient with ventilator-associated pneumonia, respectively. P. aeruginosa strains were maintained in −80°C stocks until required. Prior to use in cell culture, each strain was grown to mid-log phase in Luria-Bertani (LB) broth (Invitrogen) and bacterial concentrations at an optical density at 600 nm (OD₆₀₀) of between 0.4 and 0.6 were quantified by serial dilution and plating to enumerate CFU (GeneQuant Pro spectrophotometer; Amersham Biosciences). Heat-killed P. aeruginosa preparations were produced by heating a known concentration of P. aeruginosa in PBS to 95°C for 10 min.

The Staphylococcus aureus strain used in this study was an antibiotic-sensitive Newman clinical isolate [57 (link)]. SA was expanded by incubation in tryptic soy broth (TSB, Waltham, MA, USA) at 37 °C and 200 rpm, and harvested on the day of the infection experiments (see below) when an optical density at 600 nm of 0.6 was achieved (i.e., start of the exponential growth phase), corresponding to 1 × 10⁸ CFU (colony-forming unit)/mL. Optical densities were measured using a GeneQuant pro spectrophotometer (Amersham Biosciences, Little Chalfont, UK) and CFUs counted as detailed below. Prior to infection, bacteria were resuspended with the alpha minimum essential medium containing ribonucleosides, deoxyribonucleosides, 2 mM L-glutamine, and 1 mM sodium pyruvate (α-MEM, Waltham, MA, USA), supplemented with 10% FBS in the absence of penicillin/streptomycin (P/S) and ascorbic acid. In some experiments, SA was inactivated by overnight incubation with 2% (w/v) paraformaldehyde, followed by quenching with 50 mM NH₄Cl, then washed, and resuspended with phosphate buffered saline (PBS).