Cytokine and Chemokine Profiling of Activated Endothelial Cells

Cytokine and chemokine release from LPS-activated endothelial cells was analyzed using the Proteome Profiler Human Cytokine Array (Panel A) (R&D Systems, Minneapolis, USA), simultaneously detecting multiple analytes in cell culture supernatants. The applied panel allowed the identification of 13 substances, i.e., MIF (macrophage migration inhibitory factor), interleukin (IL)-8, Serpin E1, GM-CSF (granulocyte-macrophage colony-stimulating factor), GROα (growth-regulated oncogene α), IL-1α, IL-1β, IL-1ra, IL-6, MCP-1 (monocyte chemoattractant protein-1), MIP-1α (macrophage inflammatory protein 1α), RANTES (C-C motif chemokine ligand 5/regulated on activation, normal T cell expressed and secreted), and TNF-α (tumor necrosis factor alpha). The assay was performed using a cell culture medium, derived from HUVECs (1 × 10⁶ cells on a 6 cm dish), treated for 16 h with the tested substances (extracts or stilbenes), at the selected concentration of 5 µg/mL, followed by 4 h stimulation of the cells with LPS (1 µg/mL). Cell culture supernatants were collected after incubation, centrifuged, and mixed with the biotinylated detection antibody cocktail provided by the manufacturer. The samples were then incubated overnight with the membrane of the cytokine assay kit. After washing off the unbound material, the streptavidin–horseradish peroxidase conjugate and a chemiluminescent cytokine quantification reagent were added. Measurements were performed using a reader Syngen Biotech Azure 300.