Comprehensive Cell Line Culture Protocol

Cell lines were obtained from the American Type Culture Collection (ATCC) and cultured according to ATCC recommendations. Briefly, T47D (ERα+/ERβ+) and triple negative (ERα-negative, PR-negative, HER2-negative) cell lines HCC 1806, MDA-231, HCC 38, HCC 1937, and BT549 [21 (link)] were cultured in RPMI 1640 media (ATCC) and with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 100,000 units penicillin and 100,000 units streptomycin sulfate (Cellgro, Manassas, VA, USA). Cultures were maintained at 37 °C in a 5% CO₂ atmosphere using a tissue culture incubator. For estrogen-free conditions, cells were maintained in phenol red-free RPMI media with dextran-coated, charcoal-treated (DCC) FBS (cFBS).