Ar 1 mp confocal laser microscope

LysoTracker Red DND-99 (Thermo Fisher Scientific, Cat. #L7528) was used for labeling the intracellular organelles of the cells to observe the subcellular localization of Aβ^pH sensors inside the cells after phagocytosis. 20 000 cells/250 μL were plated in 14 mm microwells of 35 mm glass bottom dishes (MatTek #P35G-1.5-14-C) and kept overnight. The cells were treated with 5.0 μM Aβ^pH for 2 hours on the next day. Then the Aβ^pH-containing medium was aspirated and replaced with 200 μL of media containing 100 nM concentration of the LysoTracker dye and the cells were incubated in a 37 °C, 5% CO₂ incubator for 30 minutes. Finally, the cells were fixed and treated with DAPI to stain the nuclei followed by 2–3 drops of ProLong Gold Antifade Mountant using the above-mentioned protocol. Fluorescence images of phagocytic microglial cells were captured using 40× and 60× objectives on a Nikon AR-1 MP confocal laser microscope. Images were obtained using the NIS Elements microscope imaging software.

For labeling the cells with phalloidin and DAPI, 20 000 cells/250 μL were plated in 14 mm microwells of 35 mm glass bottom dishes (MatTek #P35G-1.5-14-C) and kept overnight. The cells were treated with 5.0 μM Aβ^pH for 2 hours on the next day. Then the medium was aspirated, and cells were fixed with 4% paraformaldehyde for 20 minutes, and then gently washed once with PBS. Phalloidin-iFluor 594 reagent (Abcam, Cat. #ab176757; 1000× stock) was diluted to 1× in PBS and added to the fixed cells for 10 minutes for staining the actin filaments. To label the nuclei, DAPI was diluted to a concentration of 1 μg mL⁻¹. The cells were washed again with PBS followed by a 10 minute incubation with the diluted DAPI solution. Finally, the DAPI solution (Invitrogen, Cat. #D3571) was aspirated and the fixed cells treated with 2–3 drops of ProLong Gold Antifade Mountant (Invitrogen #P36930) before imaging. Fluorescence images of phagocytic microglial cells were captured using 40× and 60× objectives on a Nikon AR-1 MP confocal laser microscope. Images were obtained using the NIS Elements microscope imaging software.