“…For co-culture assays, we seeded 12,000 total cells per cm 2 in 35 mm dishes with a ratio of 1:9 ER+ breast cancer cells to MSCs. One day after seeding, we washed cells once with phosphate buffered saline (PBS) and then changed medium to phenol-red free DMEM (#A14430-01, Gibco), supplemented with 1% FBS, 1% P/S, 1% Glutamax, 1% sodium pyruvate, 10 nM β-estradiol (#E2758, Sigma-Aldrich, Millipore Sigma, Saint Louis, MO, USA), and 5 mM glucose (89). We cultured cells in low serum (1%), low glucose (4 mM), and 10 nM estrogen (LG/LF) medium for 3 days before analyzing experimental endpoints.…”