Pharmingen fitc annexin 5 apoptosis detection kit 2

Analysis of cell cycle phases was performed by propidium iodide (PI) (Sigma-Aldrich) staining. Cells were transfected with siRNAs equivalently to the proliferation assays (see above), harvested after 96 h (including supernatant), fixed in ethanol (70%) at 4 °C, and stained with PI solution (50 µg ml⁻¹, with 20 µg ml⁻¹ RNAse A (Invitrogen)). Analysis of apoptosis has been performed by combined Annexin V-FITC/PI staining (BD Pharmingen FITC Annexin V Apoptosis Detection Kit II; BD Biosciences). Cells were transfected with siRNAs equivalently to the proliferation assays (see above) and harvested after 96 h (including supernatant). The samples were assayed on an Accuri C6 flow cytometer and analyzed with the Accuri C6 CFlow Plus software. An example of the gating strategy is given in Supplementary Fig. 6.

MDA-MB-231, MDA-MB-468, SUM159, MCF7, BT-474, SkBr-3 and WT iMMECs were incubated with 15 μM of DMSO or 3 μM KPT-8752 or 1 μM KPT-9274 for 72 h. Apoptosis was assessed by staining with Annexin V and propidium iodide. Annexin V is a membrane phosphatidylserine (PS) binding protein. It binds to the cells early in apoptosis, which is characterized by PS being flipped to face the outer membrane of the cells. Propidium iodide can enter the cell and bind to nucleic acid, but only after the membrane has begun to rupture, a characteristic of more advanced apoptosis. To assess binding by Annexin V and propidium iodide, cells were trypsinized into single cell suspension, counted, washed with 1X Annexin V binding buffer and stained with Annexin V and Propidium Iodide (BD Pharmingen FITC Annexin V Apoptosis Detection Kit II, BD Biosciences, Franklin Lakes, NJ). The cells (1 × 10⁵) were incubated with Annexin V and Propidium iodide for 15 minutes in the dark at room temperature, then washed with 1X Annexin V binding buffer and analyzed by flow cytometry using a Gallios Cytometer (Applied Biosystems, Foster City, CA).