Dichlorofluorescein diacetate

Cells were plated at 50,000 cells per well in 96-well plates, protected or unprotected by 10 µM ascorbic acid. Following a 4 h incubation period, the cells were then treated with the indicated compounds at the IC₅₀ or with H₂O₂ as a positive control. Twenty-four hours later, the plates were centrifuged and the supernatants were collected. The adherent cells were treated with 20 µL of 0.25% trypsin (Invitrogen) for 2 min. The cells were then removed and transferred to black-walled 96-well plates to prevent bleeding of fluorescence between sample wells. Dichlorofluorescein-Diacetate (10 µM) (Invitrogen - Molecular Probes), was then added to each well. The plates were then immediately read on a fluorescent plate reader using excitation wavelength of 485 nm and emission wavelength of 538 nm. Fluorescence was determined every 10 min over a 320 min. period.

To detect intracellular ROS levels, cells were incubated with 1 μM dichlorofluorescein diacetate (Invitrogen) for 30 min and washed with warm phosphate-buffered saline (PBS). Mitochondria-specific ROS were detected using MitoSOX Red (5 μM for 15 min at 37 °C) following the manufacturer’s protocols (Invitrogen). For all mitochondrial assays, cells were treated with vehicle, 1 μM Aβ, or 500 ng/ml TSP-1 for 24 h, or pretreated with TSP-1 for 1 h and co-incubated with either 1 μM Aβ or DMSO for the next 23 h. Fluorescent signals were captured using a fluorescence microscope (Olympus), and >100 cells were analyzed for each group.