Quantikine human il 6

Human CD14⁺ monocytes were isolated from the blood of a healthy donor (67 years old man). The blood sample collection was approved by the Ethics committee of the EFS Blood Bank Center. After the mononuclear cells’ isolation by the Ficoll gradient centrifugation method, the CD14⁺ cells were selected by positive selection thanks to magnetic beads (MACS^® Miltenyi Biotec B.V. and Co. KG, Bergisch Gladbach, Germany). Monocytes were then cultivated for 6 days in 24-well plates, with α-MEM medium supplemented with 10% fetal bovine serum (FBS) and 100 ng/mL macrophage colony-stimulating factor (M-CSF) (R&D Systems, Inc., Minneapolis, MN, USA, Ref: #216-MC). After these 6 days in culture, the medium was replaced with α-MEM supplemented with 10% FBS and 50 ng/mL M-CSF in addition to 100 ng/mL LPS (Sigma, Ref: #L4391) and either the Veh. or c-MIM-3. The treatments were applied for 24 hours and each condition was performed in triplicate. At the end of the incubation period, SN were collected, aliquoted, and stored at −80°C until ELISA assay (human IL-6 Quantikine, R&D Systems). Optic density was measured on a spectrophotometer Multiskan Go and the Skanlt 3.2 software was used to quantify the measured cytokine concentrations.