0.22 μm syringe driven filter

The S. aureus Newman WT harboring the complementation vector pDC123, the blaI mutant + pDC123 and the complemented blaI mutant strains were grown in THB, Cm 10 μg/mL, washed and adjusted to equal CFU concentrations in PBS. Subsequently, 10 μL of the bacterial suspensions were dropped onto THA skim milk plates containing 10 μg/mL Cm to compare the proteolytic activities. The agar plates were incubated for 24–72 h at 37°C. Areas of clearance around the colonies indicated secreted protease activity. Protease activity tests with the MRSA252 WT and blaI mutant strains were carried out using skim milk agar plates without Cm.
To assess cathelicidin degradation, supernatants from staphylococcal overnight cultures grown in TSB were filter sterilized through a 0.22 μm syringe-driven filter (EMD Millipore). 18 μl of filtered supernatants were incubated with 2 μl of CRAMP or LL-37 to give a final concentration of 16 μM CRAMP or 8 μM LL-37. Samples were incubated at 37°C for 24 h, mixed with 4x sample buffer and 10x reducing agent (Life Technologies), boiled for 10 min, loaded onto a 12% Bis-Tris gel (Life Technologies) and run at 120 V in MES running buffer. Gels were stained with SimplyBlue SafeStain (Life Technologies) and subsequently destained in H₂O.

Experiments were performed in adult male Sprague Dawley (SD) rats obtained from Charles River (UK). The body weight range at the start of the experiments was 210–280 g. Food and water were provided ad libitum. Their body weight and general aspects of health were monitored daily.
Freshly-made cobalt chloride hexahydrate (CoCl₂.6H₂O; Sigma-Aldrich, Dorset, UK) solutions dissolved in distilled water (dH₂O) and dH₂O were sterilised through a 0.22 μm syringe-driven filter (Merck Millipore, Watford, UK).
Two separate in vivo experiments were performed successively. The first experiment was a time-response experiment in which a 7-day cobalt treatment was compared against a 28-day treatment at a fixed cobalt dose. Animals were treated daily with either 1 mL/kg body weight dH₂O (controls) or 1 mg/kg body weight (BW) CoCl₂ doses injected i.p. (treated groups). The second in vivo experiment was a dose-response experiment. The animals were given 1 mL/kg dH₂O i.p. in the case of the control group, or a range of cobalt solutions- 0.1, 0.5, or 1 mg/kg BW i.p. injections for 28 days. Figure 1 depicts the sample size and design information diagrammatically.