Unknown

The dendritic cell activation assay using THP-1 cells, also known as human Cell Line Activation Test were used for activity evaluation. THP-1 cells were cultured in complete RPMI 1640 with 25 mM HEPES buffer and 2 mM L-glutamine (Invitrogen, Germany) supplemented with 10% FBS (Biochrom AG, Germany), 1% penicillin (100 U/mL)- streptomycin (100 mkg/mL) (Biochrom AG, Germany) and 0.05 mM 2-mercaptoethanol (Invitrogen, Germany) in T150 culture flasks (TPP, Switzerland). The cells were kept in a humidified atmosphere at 37 C and 5% CO2. They were subcultured every 3–4 days with an initial cell density of 2 x 10^5 c/mL. Cells were passaged for 8 weeks to a maximum of 24 passages. For experiments, cells were seeded in 24 well plates (TPP, Switzerland) by adding 1 x 10^6 cells in 500 mkL per well. Substances were dissolved in medium (2) or DMSO (500). DMSO solved substances were further diluted in medium to obtain 2 stock solution. Final DMSO concentration on the cells did no exceed 0.2%. For each substance eight concentrations were tested in duplicates. Concentrations were chosen according to preliminary propidium iodide (PI) cytotoxicity assays. Therefore, cells were exposed to nine concentrations of 1:2 serially dilution starting at 2000 lg/mL for solid test compound or 1000 mkg/mL for liquid test compound, respectively. Assessment of viable cells was performed according to the main experiment procedures but cells were stained using propidium iodide only. The highest tested concentration in the main experiment was 1.2 CV75. The additional concentrations were obtained by a 1:1.2 dilution serie of the 1.2 CV75. Each concentration was run in duplicates and each experiment was performed minimum in two independent times. Treatment was performed by applying 500 mkL of the test substance dilution to each well for 24 h.