3.3. Nonspecific In Vitro Cytotoxicity in Mammalian Cells

Adhered J774.1 macrophages or differentiated THP-1 monocytes (1 × 10⁴ cells) were cultured in the compound-containing medium for 48 h to the compounds. Cell viability was then assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay [1]. In this analysis, MTT was added to a final concentration of 0.1 mg/mL to each well, and the culture was incubated at 37 °C for 3 h. The medium was removed, any formazan crystals dissolved in solubilization buffer (glycine (10 mM); NaCl (10 mM); EDTA pH10.5 (0.05 mM) in DMSO) and the absorbance at 560 nm determined. The effective compound concentration that inhibits cell growth by 50% (EC₅₀) was established using OriginLab8.5^® sigmoidal. All assays were performed in triplicate [35].

To assess a compound’s hemolytic activity the Red Blood Cell Lysis Assay (national blood bank) was performed. A compound-containing erythrocyte (2% (w/v)) suspension prepared in phosphate-buffered saline pH 7.4 was incubated for 24 h at 37 °C and the amount of hemoglobin released into the supernatant determined spectrophotometrically using a Varioskan TM Flash Multimode Reader (Thermo ScientificTM, MA, USA) set to a wavelength of 405 nm. The % hemolysis was determined using the follows equation: % released hemoglobin = [(A₁ − A₀)/(A_1water)] × 100, where A₁ and A₀ represent the absorbance at 405 nm of the test sample at 0 and 24 h, and A_1water the absorbance at 405 nm of water at 24 h. The experiments were performed by quintuplicate. Amphotericin B (final concentration of 1.5 µM) was used as a positive control [13].