3.7. Cytotoxicity Assay

Cancer cell lines were purchased from ATCC and grown following ATCC’s recommendations. Cell lines A549 (human lung cancer), MCF7 (human breast adenocarcinoma), and PC3 (human prostate cancer) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Lonza, Rockland, ME, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, ThermoFisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin (Lonza, Rockland, ME, USA) in a humidified atmosphere at 37 °C in 5% CO₂.

Cytotoxicity was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay [38]. Briefly, cells were seeded in 96 well plates (1 × 10⁴ cell/well) and allowed to settle for 24 h. Cells were then treated with different concentrations (100, 30, 10, 1, and 0.1 µg/mL) of the isolated erythrolides (compounds 1–7, 10–15) dissolved in ultrapure dimethyl sulfoxide (DMSO; Sigma–Aldrich, San Luis, Mo, USA) at 0.1 mg/mL. After 48 h, an MTT (ThermoFisher Scientific, Waltham, Ma, USA) solution (10 μL of a 5 mg/mL stock) in PBS was added to each well and incubated for 4 h at 37 °C in 5% CO₂. Finally, the supernatant was removed and 100 µL of saline lysis buffer was added and absorbance was measured in a Tecan Sunrise Eliza-Reader (Hombrechtikon, Switzerland) at 595 nm.