No f6765

For trypan blue exclusion assays, cells were trypsinized and mixed with trypan blue solution. Viable cells were determined based on dye exclusion and were counted using a TC20 Automated Cell Counter (Bio-Rad). CellTrace Violet proliferation assays were performed by staining cells with CellTrace Violet dye (Thermo Fisher) according to the manufacturer’s instructions and culturing cells for a total of seven days. A portion of cells were fixed with 10% formalin for 20 minutes on days 4, 7, and 10, washed with PBS, and stored in PBS at 4°C until analysis. CellTrace Violet fluorescence intensity was analyzed in the VUMC Flow Cytometry Shared Resource using the 5-laser BD LSRII. Datasets were analyzed using FlowJo software (FlowJo, LLC). For E2-dependent proliferation assays, cells were grown in charcoal-stripped FBS-containing (cs-FBS) media (Millipore Sigma, Catalog No. F6765) for 1.5 weeks prior to initiation of experiments to ensure estrogen deprivation. Cells were cultured in cs-FBS media in the presence or absence of E2 (10nM; Millipore Sigma, Catalog No. E2758) for a total of 3 days. Each day, proliferation and viability was assessed by trypan blue exclusion.