Quantifying Microglial Aβ Phagocytosis

Microglial phagocytosis was evaluated by quantifying the degree of internalization of the fluorescent HiLyte Fluor 488-labeled Aβ1-42 peptide (AnaSpec, AS-60479-01). Cells were seeded in 8-well LabTek removable chamber slides. After treatment, 1 μM HiLyte Fluor 488-labeled Aβ peptides was added and incubated for 4 h at 37 °C in a 5% CO₂ atmosphere. Then, cells were washed three times with PBS to arrest uptake, and plasma membrane/cytosol and nuclei were labeled with CellMask™ Orange stain (1:20,000; Thermo Fisher Sci., C10045) and DRAQ5™ (1:500; Thermo Fisher Sci., 62251), respectively, for 5 min. Finally, cells were fixed with 4% PFA and observed under a Leica TCS SPE confocal laser scanning microscope (Leica Microsystems) using a 63x/1.32–0.60 oil PH3 CS objective and a confocal pinhole set at 1 Airy unit. From each well, three non-overlapping images from the top, middle and bottom areas were randomly taken. The ImageJ software [34 (link)] was used to calculate the CTCF by applying the following formula: CTCF = Integrated Density − (Area of selected cell × Mean fluorescence of background readings).