2.5. Cell Proliferation by MTT Assay

The human breast adenocarcinoma cell lines MCF-7 (ATCC® HTB-22) and MDA-MB-231 (ATCC® HTB-26) and the breast epithelial cells MCF-10A (ATCC® CRL-10317) were acquired from the American Type Culture Collection (ATCC). MCF7 cells were cultured in Eagle's Minimum Essential Medium (EMEM; ATCC) and MDA-MB-231 cells were cultured in high glucose Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich), whereas MCF-10A cells were cultured in 1 : 1 mixture DMEM: F-12 medium (ATCC) supplemented with 20 ng/mL epidermal growth factor (EGF; Gibco, Thermo Fisher Scientific), 0.01 ng/mL insulin (Sigma-Aldrich), 500 ng/mL hydrocortisone (Sigma-Aldrich), and 5% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific). Tumorigenic cell lines were supplemented with 10% FCS. 1% penicillin/streptomycin mixture (Pen/Strep, 10,000 IU/ml; Sigma-Aldrich) was added in each cell culture medium to avoid a possible fungal/microbial contamination. Standard conditions were used for cell culture—37°C and humidified atmosphere containing 5% CO₂, as previously described before [34, 35].

The colorimetric microculture tetrazolium assay (MTT) was used to study the viability of MCF-7, MDA-MB-231, and MCF-10A cells in accordance with Mosmann [36]. The cells were seeded in 96-well culture plates at a cellular density of 1 × 10⁴ cells/well and allowed to attach to the bottom of the well. The cells were treated with various concentrations of the tested samples—25, 50, and 100 μM, respectively, for the standard compounds (rosmarinic acid—RA and ursolic acid—UA) and 25, 50, and 100 μg/ml, respectively, of the Melissa officinalis L. extracts (dissolved in dimethyl sulfoxide—DMSO; Sigma-Aldrich) and incubated for 24 h. The control group is represented by cells treated with DMSO—the solvent used for sample preparation. Cells were then assayed by the addition of 10 μL of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution from the MTT-based in vitro toxicology assay kit (Sigma-Aldrich). Intact mitochondrial reductase converted and precipitated MTT as blue crystals during a 3 h contact period. The precipitated crystals were dissolved in 100 μL of lysis solution provided by the manufacturer. Finally, the reduced MTT was spectrophotometrically analyzed at 570 nm, using a microplate reader (xMark Microplate Spectrophotometer, Bio-Rad). The percentage of cell viability was calculated using the formula: cell viability (%) = 100 − [(A₀ − A_s)/A₀ × 100], where A₀ = absorbance of blank sample and A_s = absorbance of tested samples.