The freshly drawn blood samples were stimulated ex vivo in whole blood cultures via lipopolysaccharides (LPS). Therefore, samples were immediately diluted 1:5 with the cell culture medium RPMI 1640 including 20 mmol HEPES and L-glutamine (Sigma-Aldrich, Hamburg, Germany) and added antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin; Sigma-Aldrich, Hamburg, Germany). The samples were seeded into 12-well microtiter plates and mixed with 10 ng/mL (final concentration) LPS from Escherichia coli (Sigma-Aldrich, Hamburg, Germany). The plates were incubated for 24 h at 37 °C without a CO2 application. The used HEPES buffer is able to stabilize the pH over 24 h. The supernatants were frozen at −80 °C until analysis.
The levels of the inflammatory markers (IL-1β, IL-6, IL-10, TNF-α, MCP-1, MMP-9) in whole blood culture supernatant were simultaneously determined using a human Magnetic Luminex Assay (Bio-Techne, Abingdon, Oxon, UK) and a Magpix Luminex instrument (Luminex Corp, Austin, TX, USA).
The levels of the inflammatory markers (IL-1β, IL-6, IL-10, TNF-α, MCP-1, MMP-9) in whole blood culture supernatant were simultaneously determined using a human Magnetic Luminex Assay (Bio-Techne, Abingdon, Oxon, UK) and a Magpix Luminex instrument (Luminex Corp, Austin, TX, USA).