Freezing medium

The pellets were cultured after a pre-plating step (Sharifiaghdas et al. 2011 (link)), in 75 cm² flasks at a seeding cell density of 1800 cells/cm², coated with Matrigel™ (BD Biosciences, The Netherlands) diluted 1:200 in DMEM culture medium containing 1% PSA, until 85–90% confluency in Advanced DMEM (Life technologies, USA) containing 20% Fetal Bovine Serum (FBS, Life technologies, USA), 10% Horse Serum (HS, Life technologies, USA), 4 mM l-Glutamin (Lonza, Germany) and 1% PSA (growth medium: GM). Subsequently, medium was aspirated and the cells were washed with PBS. Next, cells were passaged by trypsinization with 0.05% trypsin-EDTA (Life technologies, USA) for 5–8 min at 37 °C, 5% CO₂ in a humidified incubator and neutralization with TNS (Life technologies, USA). The resulting suspension was spun down at 460 g for 5 min at ambient temperature. After removal of the supernatant, cells were resuspended in freezing medium (Life technologies, USA) and stored in liquid nitrogen. Phenotype of the cells was confirmed by CD56 immunostaining and ability to form myotubes upon serum withdrawal (data not shown).

FVB/N-Tg (MMTV-PyMT)634Mul/J (PyMT) 12-weeks-old mice were used as tumour donor. Aseptically collected mammary tumours from PyMT mice were minced and immersed in cold Dulbecco's Modified Eagle's Medium (Sigma, USA). Mechanical cell dissociation was performed using Medicon disposable chambers (BD bioscience, USA). The cell suspension was then progressively filtered using Filcon filters with pore sizes of 500 μm, 200 μm and 70 μm (BD bioscience). Finally, cells were aliquoted in freezing medium (Life Technologies, USA) and stored in liquid nitrogen.