Mtt reduction

Breast cancer cells were seeded at a density of 15 000 cells/cm² in 24-well plates in RPMI-1640 supplemented with 10 % FBS. Cell proliferation was quantified by MTT reduction (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide, Sigma-Aldrich, St Louis, MO, USA). Formazan salt was dissolved in acid isopropanol and absorbance was read at 570 and 630 nm in an ELISA plate reader. Results are expressed as the increase in absorbance (570–630 nm) of cells at different days over the absorbance at day 0. Experiments were performed at least three times in triplicate.

Cell viability and death were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction (Sigma-Aldrich, St. Louis, MO, USA) and Cytotoxicity Detection Kit (LDH-assay)(Roche, Basel, Switzerland) assays, essentially as described in ref. 57 (link). The Anoikis resistance assay was performed with Corning Costar Ultra-Low attachment 24-well plates (Corning Life Science, Tewksbury, MA, USA). Briefly, 48 h post transfection, cells were plated and after another 24 h, cellular viability was assessed either manually by counting cells with loss of membrane integrity, or Trypan blue exclusion followed by automatic cell counting (Cedex XS Analyzer, Roche).

The viability of the epimastigote forms was evaluated by MTT reduction assay in the presence of ABCC-like transporter modulators and in the glutathione (GSH) biosynthesis pathway inhibitor buthionine sulfoximine (BSO, Sigma-Aldrich). Briefly, 10⁷ epimastigotes/mL in complete BHI medium were distributed in 96-well culture plates at the concentrations of 2.5, 5.0, or 7.5 mM probenecid (Sigma-Aldrich); 300, 600, or 900 μM indomethacin (Sigma-Aldrich); or 100, 200, and 300 μM MK-571 (Sigma-Aldrich). After 24 h, plates were centrifuged at 1,000 × g for 10 min and the supernatant discarded. Parasites were then incubated for 4 h at 27°C with 2.5 mg/mL MTT (Sigma-Aldrich) and 0.22 mg/mL PMS (Sigma-Aldrich) in PBS supplemented with 2 g/L glucose and 10% FBS. Plates were then centrifuged and the supernatant was discarded. Formazan crystals were dissolved in DMSO (Sigma-Aldrich) and the absorbance was measured at 570 nm on a Beckman Coulter AD340 spectrophotometer (Beckman Coulter, Brea, CA, USA).