Pen strep

MCF10A cells have previously been described and were authenticated by short tandem repeat profiling. MDA-MB-231, HEK293T and MCF7 cells were purchased from ATCC. MCF10A cells were cultured in DMEM/F12 media (Corning, New York, NY, USA) supplemented with 5% horse serum (Invitrogen, Carlsbad, CA, USA), 1% Pen/Strep, 20 ng/mL EGF (ProSpec, East Brunswick, NJ, USA), 0.5 µg/mg hydrocortisone, 100 ng/mL cholera toxin and 10 µg/mL insulin. MDA-MB-231, HEK293T and MCF7 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% Pen/Strep. All cells were cultured in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C.

Bone marrow cells were flushed out of the long bones of the mice and the cell suspension cultured in αMEM supplemented with 10% FCS, Pen/Strep and 100 ng/ml M-CSF (Prospec Bio) for three days. Next the non-adherent cells were removed by washing the cultures with PBS, and the adherent BMDMs harvested using Acutase (Sigma) or cell dissociation buffer (Enzyme-Free; PBS-based) (Gibco, Life Technologies). For osteoclast formation experiments, cells were plated in 96-well plates at 15 × 10³ cells in 100 µl medium supplemented with 25 ng/ml M-CSF and up to 100 ng/ml RANKL (a kind gift of Dr. J. Dunford, University of Oxford) per well. The culture medium was refreshed at day 2 and day 4 and the cells fixed on day 5 with 4% buffered formalin and stained for TRAP. TRAP positive cells containing three or more nuclei were counted as osteoclasts. For RNA isolation, BMDM cells were seeded in 6-well plates at 5 × 10⁵ cells per well in medium supplemented with 25 ng/ml M-CSF only (for macrophage cultures) or 25 ng/ml M-CSF plus 100 ng/ml RANKL (for osteoclast cultures) and cultured for 5 days as described above. For Western Blot analysis, BMDM cells were seeded in 6-well plates at 5 × 10⁵ cells per well in medium supplemented with 25 ng/ml M-CSF and cultured for 3 days.