Human th1 th2 11plex flowcytomix kit

ADAMTS 13 activity was assessed by a commercially available chromogenic enzyme-linked immunosorbent assay (ELISA) (TECHNOZYM^® ADAMTS-13 activity, Technoclone, Vienna, Austria), according to the manufacturer's instructions. ADAMTS-13 activity <40% was defined as deficiency by the manufacturer. Plasma vWF levels were measured using IMUBIND^® vWF ELISA kits (Sekisui Diagnostics, Stamford, CT). Cytokine profile analysis, including interleukin-4, interleukin-6, interferon-γ, and tumor necrosis factor-α, were measured simultaneously using a Human Th1/Th2 11plex FlowCytomix kit (eBioscience, Bender MedSystems, Austria) according to the manufacturer's instructions.

The DCs and PBLs from venous blood obtained from healthy donors that responded to TT-Ag, using Ficoll gradient separation after informed consent was obtained. The DCs were incubated for 2 h with 30 and 60 ng/mL of FITC-TT peptide free or encapsulated within vaccine carriers coated with various ligands. In a different set of experiments, DCs were incubated with 30 and 60 ng/mL of FITC-TT peptide, poly I:C and R848 in soluble form or encapsulated within nanovaccine. Subsequently, DCs were washed and co cultured with TT-responsive PBLs (1:10 DC/T-cell ratio). Cytokine production was measured in supernatants after 24 h using fluorescent bead immunoassay (FlowCytomix human Th1/Th2 11plex kit; Bender MedSystems GmbH). Proliferative responses were determined after culturing DCs and T cells for 4 days by adding tritiated thymidine (1 OCi [0.037 MBq]/well; MP Biomedicals, Amsterdam, The Netherlands) to the cell cultures. tritiated thymidine incorporation was measured after 16 h in a scintillation counter.

Cytokine concentrations were determined using a multiplex assay according to the manufacturer’s instructions (FlowCytomix Human Th1/Th2 11 Plex Kit, FlowCytomix Human IL17-A, IL-23 and NGF Kit, Bender MedSystems GmbH, Vienna, Austria). A FACScan was used for quantification (Beton Dickinson, Franklin Lakes, NJ, USA). Data were evaluated using the Flow Cytomix Pro 2.2 software (Bender MedSystems GmbH, Vienna, Austria). The IL-4/Interferon-gamma (IFN-γ) ratio was defined as the ratio of IL-4 [pg/ml] to IFN-γ [pg/ml].

PBMCs were co-incubated with B. burgdorferi for 20 hrs. Cell-free culture supernatants were prepared by removal of PBMCs at 300×g for 10 min followed by centrifugation of the supernatant at 9000×g for 10 min to remove non-adherent spirochetes and cellular debris. Cell-free supernatants were aliquoted and stored at −20°C until further use. Concentrations of human IFN-α and IFN–λ1 were quantitated using the Human Verikine IFN-α (PBL Biomedical Laboratories) or the Human IL-29 ELISA Ready-Set Go! Kit (eBioscience), respectively. Protein levels of IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 p70, TNF-α and TNF-β in cell-free culture supernatants were measured using the FlowCytomix Human Th1/Th2 11plex Kit (BMS810FF;Bender MedSystems) according to the manufacturer’s instructions. Data were acquired with a MACSQuant analyzer (Miltenyi Biotec) and analyzed using FlowCytomix Pro Software (Bender MedSystems).