Magpix luminex platform

Interferon alpha (IFN-alpha), transforming growth factor alpha (TGF-alpha), interleukin (IL)-4, IL-6, IL-10, IL-12, and monocyte chemoattractant protein (MCP)-1 concentrations were measured in the supernatants of Hofbauer cells infected with rrRSV at MOI of 1.0 and uninfected controls after removing cellular debris by centrifugation. All samples were analyzed at 24-hour post-infection using a Milliplex MAP Human Cytokine/Chemokine Magnetic Cytokine kit and the Magpix/Luminex platform in a multiplex format (EMD-Millipore, Burlington, MA). Tumor necrosis factor alpha (TNF-alpha) was analyzed using conventional sandwich ELISA kits (Thermo Fisher).

Protein release from OA tissues and stimulated SFs into the CM was evaluated using multiplex bead-based sandwich immunoassay kits. Concentrations of 11 biomolecules (EGF, Eotaxin, FGF-2, IL-10, IL-1Ra, IL-8, IP-10, MCP-1, PDGF-AB/BB, RANTES, VEGF) were quantified at least in duplicate for each sample using MILLIPLEX^® Assays (Merck KGaA, Gernsheim, Germany) according to the manufacturer’s protocol and the MAGPIX Luminex platform. Briefly, 25 µL of the standards, controls and samples was added to each well, and then 25 µL of antibody-immobilized beads was added. The plate was incubated overnight at 4 °C, then washed with 200 µL washing buffer and incubated with 25 µL of detection antibodies for 1 h at RT. Finally, the plate was incubated with 25 µL of streptavidin-PE for 30 min at RT, washed and measured in a reader. Next, xPONENT software 4.2 for MAGPIX (Luminex Corporation, Austin, TX) and Bio-Plex Manager 6.1 (Bio-Rad Laboratories, Hercules, CA, USA) were used for data analysis. Once standard curves were generated, concentrations were interpolated for each sample using a 5-parameter curve fitting equation and expressed in pg/mL.