Fluorokine map human base kit a

Supernatants of inferior turbinate tissue and PBMC solutions were separated by centrifugation; aliquots of the supernatants were snap frozen and stored immediately at -20°C until analysis of cytokines. Concentrations of IL-1β, IL-2, IL-5, IL-6, IL-10 and IL-17, TNFα and/or interferon-γ (IFN-γ) (detection limits 0.6 to 7.8 pg/ml) were measured using commercially available Fluorokine MAP Human Cytokine Kits by using the Fluorokine MAP Human Base Kit A (R&D Systems, MN, USA) following the instructions of the manufacturer, on a Bio-Plex 200 Array Reader (Bio-Rad, Hercules, CA, USA).

PBMC were thawed and re-suspended in RPMI-1640 medium supplemented with 10% human AB serum (Biochrom, Merck, Germany), 2mM L-glutamine (PAA), 50 µM 2-mercaptoethanol (Sigma Aldrich, St. Louis, MO, USA) and 0.1 mg/ml gentamycin (Sigma Aldrich). For re-stimulation, PBMC (8 × 10⁵/well) were incubated in a 96-well plate with the JEV antigen (12.5 µg/ml), TBE antigen (2.4 µg/ml), superantigen staphylococcal enterotoxin B (SEB; 1 µg/ml) or just medium in a final volume of 200 µl. The JEV antigen was kindly provided by Dr Klade, formerly of Intercell and now Valneva. The TBE antigen strain Neudörfl was provided by the company that was Baxter Innovation, Vienna, Austria and is now Pfizer. Plates were incubated at 37 °C and 5% of carbon dioxide for 48 hours. Thereafter, supernatants were harvested and stored at −20 °C until they were tested during the following days.
The quantification of IL-2, IFN-γ and IL-10 levels were performed by Luminex technology, according to the protocols, using the Fluorokine MAP Human Base Kit A (R&D Systems, Minneapolis, MN, USA) and a Luminex reader (AtheNA Multi-Lyte®, Zeus Scientific, Raritan, NJ, USA).

The immunomodulatory effect of D. salina biomass was assessed according to the method described by Hyrslova et al. [19 (link)], with some modifications. Eight samples of blood from healthy adults were ordered from the Blood Transfusion Center of General Faculty Hospital (Prague, Czech Republic) for the isolation of human peripheral blood mononuclear cells (hPBMCs) via Ficoll–Hypaque gradient separation. Following separation and purification, the final concentration of hPBMCs was adjusted to 10⁷ cells/mL. Mononuclear cells (0.1 mL) were stimulated in an X-vivo medium (Cambrex, East Rutherford, NJ, USA) with 0.1 mL of 0.5%, 1.0%, or 3.0% aqueous solution of D. salina biomass at 37 °C for 3 days. The total volume of the solution was 1 mL. The concentrations of selected cytokines after hPBMC stimulation with aqueous solutions of D. salina biomass were evaluated using a Fluorokine MAP Human Base Kit A (R&D Systems, Minneapolis, MN, USA) for interferon (IFN)-γ, interleukin (IL)-4, IL-6, IL-10, IL-17, and tumor necrosis factor (TNF)-α, by multiplex analysis using a Luminex 200 Analyzer (Luminex Corp., Austin, TX, USA). The concentration of cytokines produced by hPBMCs was assessed using Luminex IS 2.3 (Luminex Corp., Austin, TX, USA). The results were obtained from three independent measurements.