4.8. Cell Proliferation Assay

The effect of the σ2R/TMEM97 ligands on the proliferation of the breast cancer MDA-MB-231 and MDA-MB-468 cell lines was evaluated using the BrdU cell proliferation assay kit and following the manufacture’s protocol. Briefly, MDA-MB-231 cells were shed in a 96-well plate and grown in the presence of the vehicle, PB28, SM21 or the combination of SM21 + PB28. Alternatively, in order to demonstrate the efficiency of the antiproliferative effect of NO1 independently of the genetic background of triple-negative breast cancer cells, we also used the MDA-MB-468 cell line, which was incubated with NO1 under similar experimental conditions to those used in MDA-MB-231 cells. After 24 and 48 h from the beginning of the experiment, cells were incubated for 2 h at 37 °C with a 5-bromo-deoxyuridine (BrdU) solution that was naturally incorporated by the cells. Once the BrdU incubation time was over, the cells were mixed for 30 min at room temperature with the fixing/denaturing solution provided with the proliferation kit. The incorporated BrdU was analyzed using a specific anti-BrdU antibody and a HRP-secondary antibody. The TMB substrate was added to each well and incubated for 30 min at room temperature. The reaction was then stopped and the TMB absorbance was immediately recorded at 450 nm using an TECAN M200 Infinite ELISA plate reader (Tecan Trading AG, Switzerland).