Pen strep

A375 (human melanoma; CRL-1619™; ATCC) and MDA-MB-231 (human breast carcinoma; HTB26™; ATCC) cells were seeded onto a 96-well culture plate at a cellular density of 6000 cells/well and attached to the bottom of the well overnight. After 24 hours, 100 μL of new medium containing Dulbecco's Modified Eagle's Medium (DMEM; Gibco BRL, Invitrogen, Carlsbad, CA, USA) and 50 μg·mL⁻¹ and 150 μg·mL⁻¹ of the tested extracts (dissolved in dimethyl sulfoxide (DMSO); Sigma-Aldrich Company) were added and incubated for 72 h; the medium was supplemented with 10% fetal calf serum (FCS; PromoCell, Heidelberg, Germany) and 1% penicillin/streptomycin mixture (Pen/Strep, 10,000 IU/mL; PromoCell, Heidelberg, Germany). The cells were then assayed by the addition of 10 μL of 5 μg·mL⁻¹ MTT solution from the MTT-based in vitro toxicology assay kit (Tox-1; Sigma-Aldrich Company) during a 4 h contact period. The precipitated crystals were dissolved in 100 μL of lysis solution provided by the manufacturer (Sigma-Aldrich Company). Finally, the reduced MTT was spectrophotometrically analyzed at 570 nm, using a microplate reader (Bio-Rad, Hercules, CA, USA). All in vitro experiments were carried out on two microplates in quadruplicates for each tested substance as well as controls.

The A375 cell line (ATCC, Manassas, VA, USA) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco BRL, Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal calf serum (FCS; PromoCell, Heidelberg, Germany) and 1% penicillin/streptomycin mixture (Pen/Strep, 10,000 IU/mL; PromoCell, Heidelberg, Germany), as previously described [40 (link)]. Melanoma cells were passaged at confluence after treatment with 5 mM Ethylenediaminetetraacetic acid (EDTA).

Normal human mesenchymal stem cells (MSCs) were obtained from the bone marrow (BMMSCs) of eight healthy orthopaedic patients undergoing hip replacement surgery. Approximately 10 ml of bone marrow were placed in culture plates, and the fibroblastic-like, plastic adherent fraction was isolated following multiple passages and was used in our experiments. The BMMSCs were further cultured and expanded in α-minimum essential medium (Gibco BRL, Invitrogen, Carlsbad, CA, United States), supplemented with 10% fetal calf serum (FCS; PromoCell, Heidelberg, Germany) and 2% penicillin/streptomycin mixture (Pen/Strep, 10,000 IU/ml; PromoCell), by incubation at 37°C in 5% CO₂ atmosphere. Medium replacement was performed every third day and, upon reaching 80% to 90% confluence, the cells were passed using 0.25% trypsin-EDTA (ethylenediaminetetraacetic acid) solution (Sigma, St. Louis, MO, United States) followed by centrifugation (10 mins, 300 ×g) and were replated in T75 culture flasks at a density of 10000 cells/cm² to ensure optimal proliferation.

Tumor cells from SK-BR-3 cell line (ATCC^® HTB-30™, Lomianki, Poland) were cultured in McCoy’s 5a medium modified (Gibco BRL, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS, PromoCell, Heidelberg, Germany) and 1% penicillin/streptomycin antibiotic solution (Pen/Strep, 10,000 IU/mL; PromoCell) using adherent culture flasks and incubated at 37 °C in 5% CO₂ atmosphere. When reaching 90% confluence, the cells were detached from the plastic surface with 0.25% (w/v) trypsin EDTA solution (Sigma-Aldrich, St. Louis, MO, USA) and further expanded at a subcultivationratio of 1:2, as recommended by the provider’s protocol.

Human melanoma cell line—A375 (ATCC^® CRL-1619^TM) was acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA); melanoma cells were grown as a monolayer in high glucose Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, Taufkirchen, Germany) combined with 10% fetal calf serum (FCS; PromoCell, Heidelberg, Germany) and 1% penicillin/streptomycin mixture (Pen/Strep, 10,000 IU/mL; PromoCell), as previously documented [89 (link)]. The cells were maintained under conventional conditions (humidified environment with 5% CO₂ and 37 °C).