Cytotoxic Effect of Compounds on THP1-Blue™ NF-κB Cells

The cytotoxic effect of the tested compounds was specified on THP1-Blue™ NF-κB cell line (Invivogen; San Diego, CA, USA), as described previously [28 (link)]. Briefly, cells resuspended in serum-free RPMI 1640 medium (Merck, Darmstadt, Germany) supplemented with antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin (Merck)) and 10% FBS (Merck) were seeded into 96-well plates (100 µL/well, i.e., 50,000 cells per each well). After 2 h, tested compounds dissolved in DMSO (1.25–20 µM) were added to the cells. The final concentration of DMSO was 0.1% (v/v) in each well. The viability analysis was performed after 24 h incubation with the tested substances using the WST-1 Cell Proliferation Reagent kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s manual. The amount of formazan formed, which corresponded to the number of metabolically active cells in the culture, was calculated as a percentage of the control cells, which were treated only with serum-free RPMI 1640 medium and were assigned as 100%. The IC₅₀ values were calculated by four-parameter logistic (4PL) analysis from obtained viability curves by GraphPad Prism 8.0.1 (San Diego, CA, USA) software. The results are summarized in Table 1 as mean ± SEM (n = 6).