Hilyte fluor 488 labeled amyloid β peptide 25 35

To assess the phagocytic activity, NEL-MG at a density of 2 × 10 5 cells/mL were seeded on a 12-mm coverslip in 24-well cell culture dishes. NEL-MG were treated with 2 μL of red fluorescent latex beads (2 μm, Sigma-Aldrich, St. Louis, MO, USA),
HiLyte TM Fluor 488-labeled amyloid β peptide 25-35 (2 μL), or pHrodo-conjugated synaptosomes for 2 h at 37 °C. HiLyte TM Fluor 488-labeled amyloid β peptide 25-35 (Anaspec, AS-633308) was prepared according to the manufacturer's protocol.
Phagocytic activity was then stopped by adding 2 mL of ice-cold PBS. The cells were washed twice with ice-cold PBS, fixed, stained with a microglial marker (IBA-1), and counterstained with DAPI. The cells were analyzed using confocal microscopy (TCS SP5, Leica) and a DeltaVision fluorescence microscopy system (Applied Precision).

To assess the phagocytic activity, NEL-MG at a density of 2 × 10 5 cells/mL were seeded on a 12-mm coverslip in 24-well cell culture dishes. NEL-MG were treated with 2 μL of red uorescent latex beads (2 μm, Sigma-Aldrich, St. Louis, MO, USA), HiLyte TM Fluor 488-labeled amyloid β peptide 25-35 (2 μL), or pHrodo-conjugated synaptosomes for 2 h at 37 °C. HiLyte TM Fluor 488-labeled amyloid β peptide 25-35 (Anaspec, AS-633308) was prepared according to the manufacturer's protocol. Phagocytic activity was then stopped by adding 2 mL of ice-cold PBS. The cells were washed twice with ice-cold PBS, xed, stained with a microglial marker (IBA-1), and counterstained with DAPI. The cells were analyzed using confocal microscopy (TCS SP5, Leica) and a DeltaVision uorescence microscopy system (Applied Precision).
2.12 Scratch wound assay NEL-MG were seeded onto 24-well plates in a 100% con uent monolayer until they were 95% con uent and were wounded by making a perpendicular scratch with a 200 μL pipette tip. The cells were replenished with fresh growth medium and wound closure was documented by photographing the same region at different times (0-6 h). The wound area was calculated as the open wound area/total area.