Bio plex human cytokine reagent kit

Conditioned media were harvested and processed for cytokine analysis in duplicate with a custom Bio-Rad Bio-Plex human cytokine reagent kit for IL-1 receptor antagonist (IL-1ra), IL-6, IL-8, IL-10, IL-12, granulocyte colony stimulating factor (G-CSF), GM-CSF, interferon-inducible protein (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein 1-α (MIP-1α or CCL3), MIP-1β (CCL4), regulated and normal T cell expressed and secreted (RANTES), TNF-α, and vascular endothelial growth factor (VEGF) according to the manufacturer's instructions (Bio-Rad, Hercules, CA). Data were acquired on the Bio-Rad Bio-Plex 200 reader equipped with a magnetic workstation and analyzed using Bio-Plex software version 6.0 (Bio-Rad). Values presenting a coefficient of variation beyond 10% were discarded before the final data analysis. Minimum levels of detection (pg/mL) were 4.89 for IL-1ra, 0.23 for IL-6, 0.58 for IL-8, 0.17 for IL-10, 0.26 for IL-12, 0.1 for G-CSF, 2.26 for GM-CSF, 1.83 for IP-10, 3.56 for MCP-1, 2.38 for MIP-1α, 2.69 for MIP-1β, 0.49 for RANTES, 8.84 for TNF-α, 3.54 for VEGF.

Conditioned media (CM) of Mel1 and Mel3 cells untreated and treated with Caff, were collected and centrifuged to remove cell debris. CM were analyzed with a magnetic-bead-based multiplex immunoassay, i.e., xMAP technology on the Luminex platform, carried out according to the manufacturer’s guidelines (Bio-Plex, Bio-Rad Laboratories, Milan, Italy). Cytokines/chemokines were measured with a custom Bio-Rad Bio-Plex human cytokine reagent kit for IP-10, monocyte chemoattractant protein-1, MIP-1α, MIP-1β, RANTES, IL-1β, IL-10, IL-12, IL-17, IL-1ra, TNF-α, and IFN-γ. Data were acquired on the Bio-Rad Bio-Plex X200 reader equipped with Bio-Plex Pro™ Wash Station and analyzed using Bio-Plex Software Manager™ 6.1 (Bio-Rad, Hercules, CA, USA).