X vivo medium

Human muscle cells from the cell line CCL136 (ATCC; American Type Culture Collection, Manassas, VA) were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum (FCS; Biochrom, Berlin, Germany), 1% penicillin/streptomycin (Biochrom, Berlin, Germany), and 1% L-glutamine (Invitrogen, Karlsruhe, Germany) at 37°C in a humidified atmosphere of 5% CO₂. CCL136 cells stably transfected with a GFP-Atg8/LC3 fusion construct were used for autophagy experiments. Cells seeded in chamber slides and culture wells were exposed to the inflammatory cytokines IFN-γ (300 U/mL) and IL-1β (20 ng/mL) in serum-free X-vivo medium (Cambrex Bio Science, Walkersville, USA) in duplicate. To regulate autophagy, chloroquine (50 μM, Sigma, St. Louis, USA), 3-methyladenine (3MA, Sigma, St. Louis, USA), and rapamycin (1 μg/mL, Sigma, St. Louis, USA) were used. For signalling pathway analysis, CCL136 cells were stimulated in the presence of the ERK/p42/44 inhibitor PD98059. Experiments were terminated after 24 or 48 hours and cells were analyzed using fluorescence microscopy.

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.