Legendplex data analysis software v7

Blood was collected using heparinized glass capillaries and transferred into heparinized tubes. After centrifugation at 500 × g for 5 min, plasma was removed and centrifuged at 2000 × g for 5 min and stored at −80 °C until cytokine analysis was performed using the LEGENDplex Mouse Multi-Analyte Flow from BioLegend (San Diego, USA) according to manufactures’ instructions. Samples were analysed with a FACS Canto II flow cytometer (BD Biosciences, Heidelberg, Germany). Data were processed using DIVA software v8.0.1 (BD Biosciences) and LEGENDplex Data Analysis Software v7.0 (BioLegend).

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 factor_EC50).
Levels of C3 and C3a in mouse EDTA plasma samples were determined using in-house ELISA described previously (22 (link), 23 (link)).