Prostate Stem Cell Labeling and BPA Exposure

Primary human prostate epithelial cells (PrEC) from disease-free organ donors were cultured in ProstaLife™ medium in the presence of $1\mathrm{\mu }\mathrm{M}$ BrdU (Sigma-Aldrich) for 10 d to label all dividing cells as previously described (Hu et al. 2017 (link)). PrEC were next transferred to 3D Matrigel culture for 5 d to permit BrdU washout during PS formation. As documented, the rapidly proliferating progenitor cells lose the BrdU label, whereas the slow-dividing stem cells retain BrdU (Hu et al. 2017 (link)). These PS cultures contained either vehicle (0.1% ethanol), 2.5, or $25\text{nM}$ BPA during BrdU washout phase ( $n=4/\text{treatment}$ ). PS were harvested by 1 U/ml dispase digestion for 15 min and allowed to attach to chamber slides during overnight culture in ProstaLife™ medium. Spheres were fixed in ice-cold acetone/methanol (1:1) for 1 h and immunostained using mouse antihuman BrdU antibody 1:200 (Sigma-Aldrich) followed by secondary antibody goat antimouse Alexa Fluor® 488 (Invitrogen) with DAPI counterstain (Vector Laboratories). $\text{<a class="product" href="/product/myThCZIBPBHhf-iF58Rz/" target="\_blank">BrdU</a>}+$ label-retaining cells were identified and counted in $>100$ spheres using fluorescent microscopy (Zeiss Axioskop 20).