Cm h2dcfda

Aβ₄₂ oligomers (1 µM monomer equivalents) were added to the cell culture medium of SH-SY5Y cells seeded on glass coverslips for 15 min, in the absence or presence of 1 µM trodusquemine. To detect intracellular ROS production, cells were then loaded with 10 µM 2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA, Life Technologies, CA, USA)^{70 (link)}. Pre-treatment experiments were carried out by incubating the cells for 15 min with 1 µM trodusquemine, after which time they were washed with PBS and subsequently exposed to oligomers for 15 min. Cells were labeled with the CM-H₂DCFDA probe in the last 10 min of treatment, and the resulting fluorescence was analyzed by a TCS SP5 scanning confocal microscopy system (Leica Microsystems, Mannheim, Germany) equipped with an argon laser source, using the 488 nm excitation line. A series of 1.0 µm thick optical sections (1024 × 1024 pixels) were taken through the cells for each sample using a Leica Plan Apo ×63 oil immersion objective (Leica Microsystems, Mannheim, Germany) and then projected as a single composite image by superimposition. The confocal microscope was set at optimal acquisition conditions, e.g., pinhole diameters, detector gain and laser powers. Settings were maintained constant for each analysis.

Cellular ROS production in GECs was determined as described previously [3,39]. Briefly, GECs were pre-stimulated with ATP [3 mM] and further incubated with 50 μM APCP. GECs were labeled with the fluorescent probe 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA) (Invitrogen) which was solubilized in dimethyl sulfoxide (Sigma) and diluted to 5 μM in Hanks balanced salt solution (HBSS) containing MgCl₂ and CaCl₂ (Invitrogen). The CM-H₂DCFDA (green) fluorescence intensity was measured using a Biotek H1 M monochromatic bottom fluorescence plate reader at excitation 495 nm and emission 525 nm. The representative live images of CM-H2DCFDA fluorescence intensity were taken using epifluorescence (DM IRE2 HC inverted scope, Leica Microsystems GmbH) microscopy equipped with DIC. The single exposure images were collected sequentially in fluorescence and DIC using a Retiga 4000 r CCD camera (Qimaging). Diphenyleneiodonium (DPI), a broad-spectrum NADPH oxidase inhibitor was used as a positive control.