Cytokine levels and concentrations of IgG1, IgG2a, IgG2b, and IgG3 in mouse EDTA plasma were measured by using murine LEGENDplex™assays (BioLegend, San Diego, CA, USA) according to the manufacturer´s instructions. FACS Canto II flow cytometer was used for measurements and the LEGENDplex™ Data Analysis Software v7.0 (BioLegend) for data analysis.
Levels of S. aureus-specific IgM were determined by ELISA as described previously (21 (link)). In brief, ELISA plates were coated overnight at 4°C with 50 μl of extracellular proteins of S. aureus NewmanΔspa (cultivated in TSB; 10 μg protein/ml 5%SDS). After blocking, mouse plasma was added in a 1:3 serial dilution (dilution range: 1:50 - 1:12,150) in blocking buffer (Blocking Reagent for ELISA; Roche, 11112589001). Goat anti-mouse-IgM POD (Jackson ImmunoResearch, West Grove, PA, USA; 0.8 mg/ml) was used as a detection antibody at a dilution of 1:2,500. Optical density was measured at 450 nm using a Tecan infinite M200PRO (software: i-control 1.10). All measurements were performed in duplicates and the means were used for analysis. Antibody binding was calculated using the non-linear standard curve protocol for GraphPad Prism (EC50 × dilution factorEC50).
Levels of C3 and C3a in mouse EDTA plasma samples were determined using in-house ELISA described previously (22 (link), 23 (link)).
Levels of S. aureus-specific IgM were determined by ELISA as described previously (21 (link)). In brief, ELISA plates were coated overnight at 4°C with 50 μl of extracellular proteins of S. aureus NewmanΔspa (cultivated in TSB; 10 μg protein/ml 5%SDS). After blocking, mouse plasma was added in a 1:3 serial dilution (dilution range: 1:50 - 1:12,150) in blocking buffer (Blocking Reagent for ELISA; Roche, 11112589001). Goat anti-mouse-IgM POD (Jackson ImmunoResearch, West Grove, PA, USA; 0.8 mg/ml) was used as a detection antibody at a dilution of 1:2,500. Optical density was measured at 450 nm using a Tecan infinite M200PRO (software: i-control 1.10). All measurements were performed in duplicates and the means were used for analysis. Antibody binding was calculated using the non-linear standard curve protocol for GraphPad Prism (EC50 × dilution factorEC50).
Levels of C3 and C3a in mouse EDTA plasma samples were determined using in-house ELISA described previously (22 (link), 23 (link)).
Free full text: Click here
