β secretase activity kit

β-secretase activity in mouse brains was determined using a commercially available β-secretase activity kit (Abcam, Inc, Cambridge, MA, USA). Solubilized membranes were extracted from brain tissues using a β-secretase extraction buffer, incubated on ice for 1 h and centrifuged at 5000×g for 10 min at 4 °C. The supernatant was then collected. A total of 50 µL of sample (total protein 100 µg) or blank (β-secretase extraction buffer 50 µL) was added to each well (used 96-well plate), followed by the addition of 50 µL of 2 × reaction buffer and 2 µL of β-secretase substrate incubated in the dark at 37 °C for 1 h. Fluorescence was read at excitation and emission wavelengths of 355 and 495 nm, respectively, using a fluorescence spectrometer (Gemini EM, Molecular Devices, California, USA).

β-Secretase activity in mice brains was determined using a commercially available β-secretase activity kit (Abcam, Inc, Cambridge, MA, USA). Protein was extracted from brain tissues (hippocampus regions) using ice-cold extraction buffer, incubated on ice for 20 min and centrifuged at 10,000×g for 5 min 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 beta-secretase substrate incubated in the dark at 37 °C for 2 h. Fluorescence was read at excitation and emission wavelengths of 355 and 510 nm, respectively, using a Fluostar Galaxy fluorimeter (BMG Lab Technologies, Offenburg, Germany) with Felix software (BMG Lab Technologies, Offenburg, Germany). Beta-secretase activity is proportional to the fluorimetric reaction and is expressed as nanomole per milligram of protein per minute.

β-secretase activity in the mice brains was determined using a commercially available β-secretase activity kit (Abcam, Inc., Cambridge, MA, USA). Solubilized membranes were extracted from hippocampus tissues using β-secretase extraction buffer, incubated on ice for 1 h, and centrifuged at 5000×g for 10 min at 4 °C. The supernatant was collected. A total of 50 μL of the sample (total protein 100 μg) or blank (β-secretase extraction buffer 50 μL) was added to each well (used 96-well plate) followed by 50 μL of 2X reaction buffer and 2 μL of β-secretase substrate incubated in the dark at 37 °C for 1 h. Fluorescence was read at excitation and emission wavelengths of 335 and 495 nm, respectively, using a fluorescence spectrometer (Gemini EM; Molecular Devices, CA, USA).

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).

β-secretase activity in the mice brains was determined using a commercially available β-secretase activity kit (Abcam, Inc., Cambridge, MA, USA). Solubilized membranes were extracted from brain tissues using β-secretase extraction buffer, incubated on ice for 1 h and centrifuged at 5000×g for 10 min at 4 °C. The supernatant was collected. A total of 50 μL of sample (total protein 100 μg) or blank (β-secretase extraction buffer 50 μL) was added to each well (used 96-well plate) followed by 50 μL of 2× reaction buffer and 2 μL of β-secretase substrate incubated in the dark at 37 °C for 1 h. Fluorescence was read at excitation and emission wavelengths of 335 and 495 nm, respectively, using a fluorescence spectrometer (Gemini EM, Molecular Devices, CA, USA).