3.8. Mammalian Cytotoxicity Determination

J-774A.1 murine macrophages cells (DSMZ ACC 170) were seeded in a microtiter plate (2 × 10⁴ cells per well) (Corning 3596 Polystyrene Flat Bottom 96 Well, Corning), infected with the different compounds at different concentrations, and diluted in complete medium (Gibco) without antibiotics. After 24 h at 37 °C, the supernatants were removed and a 10% of MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma-Aldrich) was added to determine cell viability. The formazan produced after 3 h was dissolved with acidic isopropanol, and absorbance was measured at 550 nm with a SPARK Multimode microplate reader (Tecan). CC₅₀ was calculated with GraphPad Prism 6.00 (GraphPad Software) as the concentration of compound that reduces the cell viability by 50%.