Bio plex kit

Human and murine cytokines and chemokines (IL-17A, IL-10, TNF-α, GM-CSF, IFN-γ, IL-6 and IL-4) were measured by Luminex using BioPlex kits according to manufacturer’s instructions, Bio-Rad Laboratories) and analyzed using BioPlex Manager 4 software.

Intracellular (lysate) and extracellular (growth media) concentrations of IL-1β, IL-8, and IL-1Ra, MCP-1, and VEGF were detected by the Luminex technique (Bio-Rad, Santa Rosa, CA, USA) with the commercially optimized Bio-Plex kits (pro-human cytokine group I assays; Bio-Rad, Santa Rosa, CA, USA) according to the manufacturer’s instructions. The detection limit of the assay was 0.6 pg/mL for IL-1β, 1.0 pg/mL for IL-8, 5.5 pg/mL for IL-1Ra, 1.1 pg/mL for MCP-1, and 3.1 pg/mL for VEGF. For the data presentation, the amount of each cytokine per 1 µg of protein was calculated. All experiments were repeated in triplicate, and a set of cytokines was re-analyzed at an independent time to confirm the results.

IL-6, IL-17 and IL-23 levels in whole blood were determined using Milliplex® MAP kit (KomaBiotech, Korea) according to the manufacturer’s instruction.
BioRad (Hercules, CA) Bio-Plex kits were used for cytokine assays from joint lysates. All samples were run in duplicate and were assayed for murine IL-6, IL-17 and IL-23 multi-plex bead based kits. The assays were run according to manufactures recommended procedures. In brief, all buffers and diluents were warmed to room temperature prior to use. Lyophilized cytokine standards were reconstituted first to a mater standard stock using 250ul diluted according to manufacturer’s direction. Four-fold serial dilutions of the master standard stock provided eight concentrations used to determine a standard curve. The concentration ranges of the standards were: IL-6: 7.8–8000 pg/ml; IL-17: 39–40000 pg/ml; IL-23: 342–350000 pg/ml. After completion of all steps in the assay, the plates were read in Luminex 200 system and the data analyzed using BioPlex Manager 6 software.

Levels of human cytokines/interleukins in culture supernatants were measured with commercially available BioPlex kits (Bio-Rad Laboratories, Cat#. 171-AK99MR2). The mouse CXCL1 and IL-6 concentrations were measured employing a house-made BioPlex assay with antibodies from the commercially available ELISA kits (R & D Systems, Minneapolis, MN).

Multiplex cytokines and chemokines were performed using the Bio-plex kits per manufacturer instructions (Bio-RAD; Hercules, CA).

Human cytokines and chemokines in culture supernatant were measured by Luminex using BioPlex kits according to manufacturer’s instructions, and analyzed using BioPlex Manager 4 software (Bio-Rad Laboratories, Hercules, CA, USA).