Dmem hams f12

We obtained the SK-BR3 cell line and the BT474 cell line from Cell Line Service (Eppelheim, Germany). The SK-BR3 cells were cultured in Dulbecco's MEM (Biochrom) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin at 37 °C and 5% CO₂ in a humidified chamber. BT474 cells were cultured in DMEM:Hams F12 (1:1) supplemented with L-glutamine, Insulin and FBS (Cell Lines Service, Eppelheim, Germany). The medium was renewed every 2–3 days. They were cultured until 70–80% confluence. Cell harvesting was performed with Accutase treatment for 10 min in a humidified chamber at 37 °C and 5% CO₂. Subsequently, the cells were rinsed with sterile phosphate-buffered saline (PBS) and counted using a Neubauer counting chamber. The cells were seeded at a density of 30,000–40,000 per well in culture slides (Falcon CultureSlides, Corning Life Science, New York, USA, Cat.# 354108). Mycoplasm infection was excluded with 4′,6-diamidino-2-phenylindole (DAPI) staining for each test.

HROG36 (RRID: CVCL_4U49), C6 (RRID:CVCL_0194), and A375 (RRID: CVCL_0132) cell lines were purchased from Cell Lines Service GmbH (Germany).
The cells were cultured in the specific medium (DMEM/Ham’s F12 1:1 for HROG36, DMEM for C6 and RPMI for A375) and supplemented with 10% 0.22 μm pore filter-sterilized fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO₂. Cells were grown in T75 flasks (15–20 mL of medium) until 75-95% confluency, then detached by 0.25% Trypsin-EDTA solution and used for spheroid formation and proliferation evaluation.