Viability Assays of Mammary Cells

After 48 h of culturing human mammary cell lines in CM, cell line medium, or DMEM, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in vitro toxicology assay or a lactate dehydrogenase (LDH) assay was carried out, as previously described^{11 (link)}, according to manufacturer’s instructions (Sigma). MTT and LDH absorbances were measured at 570 nm and 490 nm, respectively, on a Multiskan EX plate reader (Thermo Scientific, Vantaa, Finland) and background measurements of 690 nm were subtracted for both. Optical densities of wells treated with EqMDEC CM were compared to those treated with either DMEM or control (self-CM) in order to determine cell viability or relative LDH release. Values were expressed relative to wells treated with DMEM and to lysed wells for MTT and LDH release, respectively. For active caspase 3 immunostaining, 4% paraformaldehyde-fixed cells were washed with PBS and treated with 0.5% Triton X-100 for 10 min. Following a 30 min incubation in blocking solution (1% goat serum and 1% BSA in PBS) at RT, fixed cultures were probed with an anti-active caspase 3 primary antibody diluted 1:100 in PBS (ab4051, Abcam, Cambridge, MA) for 1 h, followed by incubation with HRP-conjugated goat anti-rabbit diluted 1:100 in PBS (Jackson ImmunoResearch, West Grove, PA) for 1 h.