Cytotoxicity assay on peritoneal macrophages using resazurin.

Peritoneal macrophages were seeded at 60,000 cells per well in 96-well plates and grown in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin-glutamine in a 5% CO₂ environment at 37°C. Drugs and metals were added at the concentrations indicated below. To determine the Cu-related toxicity of the drugs on eukaryotic cells, compounds were diluted in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin-glutamine and then transferred into the wells containing cells. The plates were incubated at 37°C in a 5% CO₂ incubator for up to 48 h. Resazurin dye was added to each well at a final concentration of ∼90 μM. After continued incubation for ∼6 h, microscopic pictures were taken (magnification, ×20) and the fluorescence of the metabolically converted resazurin dye was quantified using a Cytation3 imaging reader (BioTek) and its accompanying Gen5 software.