Cells were obtained from ATCC and grew in DMEM without phenol red, supplemented with 10% FBS and penicillin/streptomycin at 37°C in a 5% CO2 and 95% humidified atmosphere. For each assay, cells were seeded at the density of 104 cells/cm2. Before treatments, to reduce estrogen levels in FBS and avoiding any interference, cells were cultured for 24 hours in medium containing 5% dextran-coated charcoal-treated serum. Then, cells were treated for 24 hours with E2, S, raloxifene, S + raloxifene, E2 + S, and E2 + raloxifene + S. In particular, the concentration tested for S was 10 ng/ml, that for raloxifene was 2 μM, and that for E2 was 1 nM.
Cells were cultured in a 96-well plate. Cells were washed in ice-cold PBS and then fixed with 2% formalin solution in PBS for 15 min. After further washes, to prevent the nonspecific binding, cells were blocked with a 10% BSA solution in PBS for 20 min and then incubated with the Alexa Fluor 647–conjugated antibody for human ACE-2 (R&D Systems). After several washes, the plate was read at the fluorescence intensity of 668 nm using a microplate reader (Spark, Tecan). Then, to normalize the results, DAPI was added, and the fluorescence intensity at 461 nm was evaluated.
Cells were cultured in a 96-well plate. Cells were washed in ice-cold PBS and then fixed with 2% formalin solution in PBS for 15 min. After further washes, to prevent the nonspecific binding, cells were blocked with a 10% BSA solution in PBS for 20 min and then incubated with the Alexa Fluor 647–conjugated antibody for human ACE-2 (R&D Systems). After several washes, the plate was read at the fluorescence intensity of 668 nm using a microplate reader (Spark, Tecan). Then, to normalize the results, DAPI was added, and the fluorescence intensity at 461 nm was evaluated.