Hoechst stain

SH-SY5Y and glioma cells were seeded at a density of 10⁴ cells/well on 96-well plates and challenged with Aβ in presence or absence of MTZ for 16h. For the last 30 minutes, the cells were incubated in the experimental medium at 37 °C with 5 μM CellROX Deep Red (Life Technologies) and 1 μg/ml Hoechst Stain (Immunochemistry Technologies, Bloomington, MN), as described by the manufacturer, followed by washes. Fluorescence was measured using a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA).

For immunofluorescence microscopy detection of ROS, SH-SY5Y cells were seeded on 96-well plates (10⁴ cells/well), challenged with 50 μM ADan pE or ADan E for 2 or 4h, and further incubated at 37°C with CellROX Deep Red (5μM; Life Technologies) and Hoechst Stain (0.2 μg/ml; Immunochemistry Technologies, Bloomington, MN). After fixation in 4% paraformaldehyde images were acquired in a Nikon Eclipse Ti microscope and analyzed with Image J as above. Confirmation of IF results as well as a more quantitative evaluation of amyloid-mediated ROS generation was assessed through the use of the free radical indicator 2',7'-dichlorodihydrofluorescein diacetate (DCFDA, Life Technologies). SH-SY5Y cells, after identical 4h treatment with ADan pE and ADan E homologues as above, were loaded with the cell-permeant DCFDA (10 μM, 30 min), as described [40 (link)]. After cleavage of the acetate groups by intracellular estearases and oxidation by intracellular ROS, the sensor is converted to the highly fluorescent compound 2’7’-dichlorofluorescein. The generated fluorescence, proportional to the intracellular ROS, was evaluated in a Molecular Devices FlexStation 3 microtiter plate reader with Ex/Em wavelengths of 492–495 nm and 517–527 nm, respectively. Data are represented as percentage of the corresponding no-peptide controls.