ELISAStandard operating procedure
Cytokine production by DCs and T cells is assessed by ELISA assay according to the manufacturer’s instructions of ELISA kit.
After the times indicated, supernatants were collected and cytokine detection was performed. MesoScale Assay 7-spot (Meso Scale Discovery) or Luminex® Assay (Invitrogen) were used for detection of IL-1beta, IL-8, IL-6, TNFalpha, IL-10, IL-12p70, IFNg and IL-17A according to the manufacturer’s instructions.
These procedures allowed simultaneously measurement of different cytokines in the same supernatants. The multiplex assay format differs from conventional ELISA in one significant way: the multiplex capture antibody is attached to a polystyrene bead whereas the ELISA capture antibody is attached to the microplate well. The use of the suspension bead-based technology enables multiplexing. Microspheres are internally dyed with red and infrared fluorophores of differing intensities. Each bead is given a unique number, or bead region, allowing differentiation of one bead from another. Beads covalently bound to different antibodies can be mixed in the same assay, utilizing a 96-well microplate format.
At the completion of the sandwich immunoassay, beads were read, using the Bioplex® 200™ detection system, in single-file by dual lasers for classification and quantification of each analyte.
Alternatively, ELISA assay was performed according to the manufacturer’s instructions. ELISA kits were from Biosource rather than ELISA for IL-12p70 that was from R&D System and IL-17 from e-Bioscience.
In blocking experiment, after 2 hors of exposure with the inhibitor, supernatants were collected after 8 or 24 hours or for T cell cytokine detection, 5 days of incubation.