BLTN cell lines SUM1315 and MDA-MB-231 were obtained from Asterand. SUM1315 cells were seeded at 25 000 cells per mL in 75 cm2 culture dish (Falcon®) at 37°C under 5% CO2, in 15 mL Ham's F12 medium (Gibco®) supplemented with 5% decomplemented fetal calf serum, 10 mM HEPES* buffer, 20 mg/mL gentamycin, 10 ng/mL EGF and 4 μg/mL of insulin, according to the supplier's instructions [29 (link), 30 (link)]. The MDA-MB-231 cell line was seeded at 25 000 cells per mL in 75 cm2 culture dish (Falcon ®) at 37°C under 5% CO2, in 15 mL RPMI 1640 medium (Gibco®) supplemented with 10% decomplemented fetal calf serum and 20 mg/mL gentamycin, according to the supplier's instructions [29 (link), 30 (link)].
Cells having reached confluence were washed with Phosphate Buffer Saline (PBS, 1X, Sigma®) and Trypsinized (Trypsin 1X, Sigma®) for 5 min at 37°C. Cells were taken up in 12 mL of culture medium and centrifuged for 10 minutes at 250 G. The pellet was taken up in 5 mL of culture medium and the number of cells per mL was determined by a cell count using a vital stain Trypan Blue. This allowed for counting the cell dilutions necessary for seeding cells in varying concentrations and defined for each experiment.
Cells having reached confluence were washed with Phosphate Buffer Saline (PBS, 1X, Sigma®) and Trypsinized (Trypsin 1X, Sigma®) for 5 min at 37°C. Cells were taken up in 12 mL of culture medium and centrifuged for 10 minutes at 250 G. The pellet was taken up in 5 mL of culture medium and the number of cells per mL was determined by a cell count using a vital stain Trypan Blue. This allowed for counting the cell dilutions necessary for seeding cells in varying concentrations and defined for each experiment.