Bacterial adhesion was measured by a modified ELISA where MDMs (2.5 × 105 MDMs per well) were cultured on 96-well flat bottom tissue culture plates, washed with PBS, fixed with glutaraldehyde and incubated with biotinylated bacterial suspensions (Fallgren et al., 2001). Briefly, equal volumes of bacteria and biotin (EZ-Link-Sulfo-NHS-LC-biotin; Boule Nordic AB, Sweden) find more solution (0.2 mg mL−1 in PBS, pH 7.6) were
incubated for 2 h at ambient temperature. Staphylococcal cells were washed three times with PBS and resuspended in PBS containing 1% BSA at the original concentration. Plates containing macrophage monolayers were blocked for 1 h at 37 °C with 3% BSA in PBS. Wells were emptied, washed twice with PBS, then filled with 100 μL biotinylated bacterial cell suspension in PBS (1 × 108 CFU mL−1) and incubated for 1 h at 37 °C. Unbound bacteria were removed with PBS containing 0.05% Tween 20. NeutrAvidin™-HRP labelled (Boule Nordic AB) (100 μL of 1/1500 dilution in PBS containing 1% BSA) was added to each well and incubated for 1 h at 37 °C. Plates were washed three times with PBS, and 100-μL ImmunoPure TMB substrate kit (Boule Thiazovivin clinical trial Nordic AB)
was added to each well. Colour was allowed to develop for 5 min, and the reaction was stopped with 1 M H2SO4. The absorbance at 450 nm was measured with a microtiter plate reader. Controls used in the ELISA assay were the following: (1) wells containing cells incubated with all ingredients except biotinylated bacteria and (2) wells containing cells incubated only with ImmunoPure TMB substrate. In all experiments, four wells for each type of bacteria/cell interaction were used. Estimation of the number of attached bacteria was standardized as follows: serial 1 : 2 dilutions of biotinylated bacteria (7.8 × 104 to 1 × 107 CFU per well) in distilled water were seeded onto the bottom of 96-well plates by allowing bacteria to dry overnight at 37 °C and were then fixed for 10 min with methanol. Florfenicol ELISA with NeutrAvidin-HRP labelled and ImmunoPure TMB substrate was performed as previously described. OD450 nm values were plotted
as a function of the number of bacteria in each well. A standard curve prepared for each experiment was used to calculate the number of bacteria attached per well (Fig. 1). Phagocytosis experiments were performed by co-incubation of cells (2.5 × 105) with bacteria at 1 : 10 ratio for 20, 40, 60, 90 and 120 min. At each time point, 20 μL lysostaphin (1 mg mL−1) was added for 10 min, to lyse extracellular bacteria. Aforementioned lysostaphin concentration was the minimum concentration required to accomplish lysis of biofilm phase bacteria. Cells were washed three times with PBS and lysed by 0.1% Triton X-100. Viable intracellular bacteria were counted by plating serial dilutions of lysates on blood agar plates.