Live caspase 3 7 green detection reagent

Cells were placed in 96-well plates (2500–3000 cells/well, quadruplicate) and MTT (562nm; Millipore-Sigma #M2128) or MTS (DU4475 and MDA-MB-453 only; 492nm; Promega #G3580) survival/growth assays performed according to manufacturer’s protocol at 24hr intervals (kinetic analyses) or 48hr (dose responses) after adding compounds. Annexin-V positivity (Annexin-V Apoptosis Detection Kit; BD-Biosciences #556547), Caspase-3 activity (Caspase-3 Assay Kit; BD-Biosciences #556485), and Caspase-3/7 activity (Live Caspase-3/7 Green Detection Reagent; Invitrogen #C10723) were measured according to manufacturer’s instructions. Cell viability was determined by Trypan Blue dye (Gibco #15250061) exclusion. Propidium iodide (Millipore-Sigma #P4170) marked apoptotic subG1 DNA content, as we previously reported (77 (link)). FlowJo (BD-Biosciences; RRID:SCR_008520) was used for flow cytometric analyses.

Mammospheres were established and maintained as previously described (79 (link)). Cells were placed in low-attachment 24-well plates (1,000 cells/well) for formation assays or 96-well plates (200 cells/well) for survival/ATP production assays (CellTiter-Glo 3D Cell Viability Assay, Promega #G9681) and Caspase-3/7 activity assays (Live Caspase-3/7 Green Detection Reagent, Invitrogen #C10723). IncuCyte Live-Cell Analysis System (Sartorius) was used for real-time quantitative live-cell imaging of Caspase-3/7 activity. Luminescence (survival/ATP production) and fluorescence (Caspase-3/7 activity) were measured and representative images captured using the Cytation-5 Cell Imaging Multimode Reader (BioTek). Mammosphere (≥50 cells) number and area were determined using the Gen5 image analysis tool on the Cytation-5.