Mda mb 231

The MCF-7 and MDA-MB-231 human breast adenocarcinoma cell lines and T47D human ductal breast carcinoma cell line were purchased from the European Collection of Cell Cultures (ECACC). The cells were cultured in Dulbecco’s modified Eagle’s medium with (MDA-MB-231) or without (MCF-7 and T47D) phenol red (DMEM, Lonza, Belgium), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Lonza), 4 mM L-glutamine (Lonza), 1% penicillin/streptomycin solution (Lonza) and 10 mM HEPES buffer (Sigma, Germany). The medium without phenol red was used for the two cells lines expressing the estrogen receptor (i.e., MCF-7 and T47D) due to the potential influence of phenol red on estrogen signaling [40] (link). The cells were cultured in 75 cm² tissue culture flasks (TPP, Switzerland) at 37°C in a humidified atmosphere containing 5% CO₂. Sub-confluent cells were sub-cultured every 3–4 days. For proliferation experiments, the cells were seeded in 96-well plates (TPP) at a density of 5,000 cells/well 24 h prior to the addition of each agent or their combinations. For flow cytometric analyses, the MCF-7 cells were seeded in 60-mm Petri dishes (TPP) at a density of 210,000 cells/well. For microscopic assessments, the MCF-7 cells were seeded in 12-well plates (TPP) at a density of 35,000 cells/well.

The human gynecological cancer cell lines isolated from breast cancers (MCF7 and MDA-MB-231), and cervical adenocarcinoma (HeLa) were obtained from The European Collection of Cell Cultures, Salisbury, U.K., while the cervical carcinoma (SiHa) was purchased from the American Type Tissue Culture Collection, Manassas, Virginia, USA. All the cells were maintained in Minimum Essential Medium that was supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin at 37 °C in a humidified atmosphere condition. All media and supplements were purchased from Lonza Group Ltd. (Basel, Switzerland).

Human gynecological cancer cell lines
(MDA-MB-231, MCF-7, and HeLa) and the human glioblastoma U-87 MG cell
line were purchased from the ECACC (European Collection of Cell Cultures,
Salisbury, UK). Cells were grown in Eagle’s Modified Essential
Medium supplemented with 10% heat-inactivated fetal bovine serum,
1% nonessential amino acids, and 1% penicillin–streptomycin–amphotericin
B mixture in a humidified atmosphere containing 5% CO₂ at
37 °C. To maintain the U-87 MG cell line, the basic medium was
supplemented with 1% l-glutamine and 1% sodium pyruvate.
All media and supplements were obtained from Capricorn Scientific
Ltd. (Ebsdorfergrund, Germany). Cells in the near-confluent phase
of growth were used for the assays described below.

As a model in vitro system for cellular experiments we chosen two human breast cancer cell lines MCF-7 and MDA-MB-231 (purchased from the European Collection of Cell Cultures and American Type Culture Collection, respectively). Cells were cultured in a cell growth medium (DMEM), supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin (all from Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Cells were maintained at 37 °C in a humidified atmosphere containing 5% of CO₂. The cells were routinely subcultured 2–3 times a week in 25 cm² cells’ culture flasks.

Breast cancer cell lines MCF7, MDA-MB-231, and MCF10A were obtained from the European Collection of Cell Cultures. The cells were cultured in standard conditions: density up to 70% confluence, 5% CO_2, and 95% air, in DMEM with proper supplements indicated previously. After a 24 h pre-incubation period, the cells were treated with R-SFN at the doses of 5 μM (for all cell lines), 10 μM (for MCF10A cells), or 20 μM (for MCF7 and MDA-MB-231 cells), which were selected based on cytotoxicity assay [8 (link)]. The incubation was continued for the subsequent 72 h. Simultaneously, control cells were treated with DMSO (vehicle) at the concentration not exceeding 0.1%.