Fluostar galaxy fluorometer

β-secretase activity in the brains was determined using commercially available β-secretase fluorescence resonance energy transfer (BACE1 FRET) assay kit (PANVERA, Madison, USA) according to the manufacturer’s protocols and as described elsewhere. This formation of fluorescence was read using a Fluostar galaxy fluorometer (excitation at 355 nm and emission at 510 nm) with Felix software (BMG Labtechnologies). β-secretase activity was expressed as nmol/(mg protein-min).

β-secretase activity in BV-2 cells and astrocytes were determined using a commercially available β-secretase activity kit (Abcam, Inc, Cambridge, Massachusetts, United States). Protein was extracted from cells using an ice-cold extraction buffer, incubated on ice for 20 minutes and centrifuged at 10,000 rpm for 5 minutes at 4°C. The supernatant was collected. A total of 50 μL of sample (total protein 100 μg) was added to each well followed by 50 μL of 2 × reaction buffer and 2 μL of β-secretase substrate incubated in the dark at 37°C for 2 hours. Fluorescence was read at excitation and emission wavelengths of 355 and 510 nm respectively, using a Fluostar Galaxy fluorometer (BMG Lab Technologies, Offenburg, Germany) with Felix software (BMG Lab Technologies, Offenburg, Germany). β-secretase activity is proportional to the fluorimetric reaction, and is expressed as nmol/mg protein per minute.

We used β-secretase activity kit (Abcam) to measure β-secretase activity in astrocytes and microglial BV-2 cells. Both cells were homogenized with ice-cold extraction buffer for 10 min and then centrifuged. The supernatant was transferred to a new tube and mixed with 50 μl lysate (25-200 g of total protein), 2 × Reaction Buffer, and 2 μl β-Secretase substrate. The mixture was then placed in a covered plate and incubated in the dark at 37°C for 1 h. In order to detect the activity of β-Secretase, fluorescence was measured using a Fluostar galaxy fluorometer (excitation at 335 nm and emission at 495 nm) and Felix software (BMG Lab technologies).