Macrophage IL-6 Response to Conditioned Media

CM biological effect on activated human macrophages was performed by assessing the IL-6 release through an ELISA assay. In detail, the human THP-1 cells were maintained in RPMI 1640 medium containing glutamine and supplemented with 10% FBS and 1 mM sodium pyruvate (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Afterwards, monocytes were differentiated into macrophages, as previously described [40 (link)]. THP-1-derived macrophages were treated with the CM diluted 1:1, with complete RPMI medium for 24 h. Thereafter, the supernatants were collected, centrifugated at 3000× g for 10 min at 4 °C, filtered using a 0.2 µm Ministart sterile filter (Sartorius, Varedo, Italy), and stored at −80 °C until usage. These cellular supernatants were used to quantify the concentrations of released IL-6 using the Human IL-6 Uncoated Invitrogen ELISA Kit assay (ThermoFisher, San Diego, CA, USA) according to the manufacturer’s directions. The plates were read at 450 nm and the sensitivity of the used ELISA assay was in the range 2–200 pg/mL.