Calf bovine serum

Cancer cell lines (4T1, 67NR, and E0771) were grown on appropriate media on 10 cm tissue culture dishes (4T1 in RPMI 1640 medium with GlutaMAX, supplemented with 10% (v/v) FBS HyClone (both Thermo Fisher Scientific, Waltham, MA, USA), 1 mM sodium pyruvate, 3.5 g/L glucose, 100 μg/mL streptomycin (all Sigma-Aldrich, Saint-Louis, MO, USA), and 100 U/mL penicillin (Polfa Tarchomin S.A., Warsaw, Poland); 67NR in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) calf bovine serum (ATCC, Manassas, VA, USA), 1× nonessential amino acids, 2.0 mM l-glutamine, 100 μg/mL streptomycin (all Sigma-Aldrich, Saint-Louis, MO, USA), and 100 U/mL penicillin (Polfa Tarchomin S.A., Warsaw, Poland); E0771 in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) FBS HyClone, 2.0 mM l-glutamine, 100 μg/mL streptomycin (all Sigma-Aldrich, Saint-Louis, MO, USA), and 100 U/mL penicillin (Polfa Tarchomin S.A., Warsaw, Poland)). The dishes were washed with PBS after the cells reached confluence, and cells were incubated for 24 h with an appropriate medium without serum. Then, CM were collected, centrifuged (7 min, 4 °C, 400× g), and applied in a 1:1 ratio (CM:NFs medium) for 72 h on NF cultures for stimulation.