Human th1 th2 11plex kit

Autologous DC of patients V-A-01 and V-A-08 were matured for 48 h with the conventional cytokine cocktail (cDC), the complete prophylactic vaccine cocktail (VAC-DC), or the separate prophylactic vaccines BCG, Typhim, or Act-HIB. cDC was loaded with KLH, gp100 peptides (10 μM gp100:280–288 + 10 μM gp100:154–162), or tyrosinase peptide (10 μM tyrosinase:369–377). 1 × 10⁴ DC were co-cultured with 5 × 10⁴ autologous cells obtained from a bronchoalveolar lavage in RPMI + 7 % human serum. Cytokine production was measured in the supernatant after 24 h by cytometric bead array (human Th1/Th2 11 plex kit, eBioscience) or standard sandwich ELISA (human IL-17 DuoSet ELISA, R&D Systems). To study T cell proliferation, cells were pulsed after 4 days with 1 μCi/well tritiated thymidine for 8 h, and incorporation of tritiated thymidine was measured with a beta-counter.

Human Th1/Th2 cytokines were determined in the supernatants of CD4⁺ T cells isolated from the spleen cells of hu-NSG mice after a 3 day stimulation by CD3/CD28 beads, and in the serum samples harvested at the time of rejection (islets alone group) or at 21 days post-transplantation (islets+T_regs group). A human Th1/Th2 11plex kit (eBiosciences) was used according to the manufacturer's protocol. Data acquisition was performed on a FACS Calibur (BD Biosciences) and then was analyzed using the BD Cytometric Bead Array software (BD Biosciences). The cytokines detected were IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, TNF-α and TNF-β.

Human cytokines in supernatants and sera were assayed using a human Th1/Th2 11plex kit (eBiosciences) according to the manufacturer's protocol. Data acquisition was performed on a FACS Calibur (BD Biosciences) and analysed using The FlowCytomix Pro 3.0 Software from eBioscience (Xiao et al., 2014).