Human ifn γ flex set

IL33 amplifies both T_H1 and T_H2 responses by activating different leukocytes, in particular NK cells [36] (link). Human PBMCs were isolated from healthy blood donors by Ficoll gradient. The blood sampling of healthy volunteers was approved by the local ethics committee (Bayerische Landesärztekammer, Munich) and subjects gave written, informed consent. The NK cells were purified from PBMC using the negative NK cell isolation kit (Miltenyi Biotec). The average purity was over 96%. For assessment of the IL1RL1-blocking functionality of the monoclonal rabbit antibodies, 1×10⁵ NK cells/well were pretreated with the rabbit antibodies or the respective isotype control antibodies at different concentrations, seeded into a 96-well flat bottom plate and incubated for 1 h at 37°C. NK cells were then stimulated with 10 ng/ml IL-33 (Peprotech) and 1 ng/ml IL-12 (Sigma-Aldrich) and incubated for 20 h in RPMI 1640 medium supplemented with 10% FCS, 1% sodium pyruvate and L-Glutamine, as well as 0.1% 2-Mercaptoethanol. After this, supernatants were harvested, centrifuged and tested for IFN-γ production. For IFN-γ quantification an in-house established ELISA or BD’s CBA flex set platform was used (human IFN-γ flex set, BD Biosciences) according to the manufacturer’s instructions using a FACS Canto II.

To detect IFNγ in supernatants, the Human IFN-γ Flex Set (560111, BD Biosciences, Franklin Lakes, New Jersey, USA) was used according to the manual for the BD CBA Human Soluble Protein Master Buffer Kit. Measurements were done on BD LSRFortessa Flow Cytometer (BD Biosciences) and FCAP Array Software. Supernatants were measured twice in duplicate. For detection of IFNα in supernatants, the VeriKine-HS Human IFN-α All Subtype ELISA kit (#41115, PBL assay science, Piscataway, New Jersey, USA) was used according to manufacturer’s protocol. For calculation of IFNα concentrations, blank optical densities were subtracted from standard and sample optical densities. Supernatants were measured twice in duplicate.