2.1. Cell Culture

The mouse melanoma B16-F10 cells exhibit in vitro a mixed morphology of spindle-shaped and epithelial-like cells. It was originally isolated from C57BL/6J mouse strain affected with melanoma. B16-F10 is a metastatic variant of B16 melanomas with high tropism for lung invasion.

The mouse RAW 264.7 cells exhibit in vitro a morphology of monocyte/macrophage cells and were originally isolated from Abelson murine leukemia virus-induced tumor of the BALB/c mouse strain.

B16-F10 murine melanoma cell line (ATCC CRL-6475) and RAW 264.7 macrophage-like cell line (ATCC TIB-71) were maintained in Dulbecco’s modified eagle’s medium (DMEM; Sigma-Aldrich, Saint Louis, MO, USA). The media formulation was supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich) and 1% penicillin/streptomycin/amphotericin B mixture (Sigma-Aldrich). Cells were grown at 37 °C under a humidified atmosphere with 5% CO₂. B16-F10 (10⁴ cells/mL) and RAW 264.7 (2 × 10⁴ cells/mL) were seeded in 96-wells plates and allowed to adhere for 24 h. Unless otherwise specified, all treatments and controls were carried out in serum-free conditions [26].