Cell death was quantified based on plasma membrane permeabilization. To this aim, cells or tissue cultures were cultured under normal serum conditions (control) and serum/trophic factor deprivation (−FCS/−B27/−HS) or double deprivation with additional removal of glucose (−FCS/−Gluc) for 24–48 h. Cultures were treated in parallel with either 25–50 nM sAPPα, 50 nM APP-E1 domain or 20 nM IGF1, as indicated. PTX (Enzo Life Sciences, 100 ng/ml) or PI3K inhibitor LY294002 (Enzo Life Sciences, 10 μM) were applied 30 min before sAPPα was added to the medium. Following collection, cells were stained with 0.8 μg/ml PI, a DNA-intercalating and fluorescent agent, to visualize dead cells that emit red light at 617 nm when excited at 535 nm. Cells were counted manually under a fluorescence microscope in three random visual fields (>150 cells) and counter-stained with Hoechst to calculate the percentage of dead cells versus the total number of visualized cells. Alternatively, cells were stained with PI and analyzed with a FACS cytometer (BD Biosciences, Heidelberg, Germany). Organotypic hippocampal slices were incubated with 0.8 μg/ml PI for 20 min at 37 °C. When applying the ADAM10 (α-secretase) inhibitor GI254023X (5 μM), slices were cultured in serum-/glucose-free medium for 48 h containing the inhibitor or its respective carrier (DMSO) as control. Round circles of identical size (Ø 500 μm) were positioned in equivalent locations within the CA1 region of each hippocampus image and all PI-stained cells were counted using ImageJ software (NIH, Bethesda, MD, USA). Cell viability assays were performed with a commercial kit (CellTiter-Glo Luminescence Assay; Promega, Mannheim, Germany) according to the manufacturer's instructions. The assay quantitates ATP levels, an indicator of metabolically active cells, photometrically with a fluorescence plate reader. Additionally, we applied the live-dead cell staining kit II from PromoKine (Heidelberg, Germany) according to the manual. Cells were simultaneously stained with green fluorescent calcein-AM (4 mM; ex/em: 495/515 nm) to detect intracellular esterase activity (viable cells) and red fluorescent ethidium homodimer-3 (2 mM; ex/em: 530/635 nm) to indicate loss of plasma membrane integrity (dead cells). Full paper
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