2.2.5. Cell Culture

MDA-MB-231 cells were cultured in F-12K medium supplemented with 100 U/mL of penicillin and 100 μg/mL of streptomycin and 5% bovine serum; 4T1 cells were cultured in RPMI-1640 supplemented with 100 U/mL of penicillin and 100 μg/mL of streptomycin and 10% bovine serum; HDMVECn (primary dermal microvascular endothelial cells) and Balb/c-5064 (mouse dermal microvascular endothelial cells) were cultured in Vascular Cell Basal Medium supplemented with Microvascular Endothelial Cell Growth Kit-BBE ATCC ® PCS-110-040; A375 (human malignant melanoma) and B16F10 (mouse malignant melanoma) were cultured in Dulbecco's Modified Eagle's Medium supplemented with 100 U/mL of penicillin and 100 μg/mL of streptomycin and 10% bovine serum.

### 2.2.6. Cell Proliferation and Apoptosis Activity

For cell viability estimation, we used the CellTiter-Blue ® Cell Viability Assay (Promega). Cells were seeded into a 96-well flat-bottomed tissue culture plate at a density of 2000 cells/well and allowed to adhere to the plate by incubating at 37 ◦C under 5% CO2 overnight. Following cell attachment, the cells were incubated with the tested CD-NHF at 5% concentrations (50 μg/mL) for 72 h. For all in vitro experiments, we used the 5% CD-NHF concentration, based on our preliminary results. Control cells were treated with phosphate-bu ffered saline (PBS), which was equivalent to the amount of PBS used as vehicle. After each of the 72 h treatment time periods, 50 μL of cell viability solution was added to each well and the plate was reincubated for 4 h before fluorescence recording using a multiplate microplate reader (FilterMax F5, Sunnyvale, CA, USA). Apoptosis was assessed using the Caspase-3/7 Assay (Promega) according to the manufacturer's indications.
