3.3. Cell Metabolic Activity (MTS Test)

The MTS test was performed in 96-well plates (Thermo Fisher Scientific, Waltham, MA, USA) as a measure of cell metabolic activity. In a first set of experiments, human monocytes (0.5 × 10⁵ cells/well) and HDPCs (0.7 × 10⁵ cells/well) were exposed to loading concentrations of 1400 W, CM544 and FAB1020 (0–400 µM). In a second set of experiments, loading concentrations of all compounds (0–200 µM) were administered on human monocytes in the presence of lipopolysaccharide (LPS) 0.25 µg/mL. The concentration of LPS (from E. coli, purchased form Merck, Darmstadt, Germany) was chosen in accordance with preliminary results (Figure S1). At the established time point (24 h), the incubation medium was harvested for further analyses and complete RPMI containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) at a concentration of 0.5 mg/mL was added to each well. Cells were incubated for 4 h at 37 °C and 5% CO₂. After that, absorbance was measured at 490 nm using a spectrophotometer (Multiscan GO, Thermo Fisher Scientific, Waltham, MA, USA). The percentage of metabolically active cells in treated cultures was calculated, setting the untreated control to 100%.