Real-time Imaging of Microglial Phagocytosis

Real-time live cell imaging of CB-iMGs was performed as previously described^{28 (link),29 (link)}. Briefly, cells were imaged on the IncuCyte ZOOM live imaging system (Essen Biosciences) while incubated at 37 °C with 5% CO₂. Synaptosomes were sonicated and labeled with pHrodo Red SE (Thermo Fisher Scientific) and added to CB-iMGs at 15 µg total protein per well in 24-well plates. Phase contrast and red fluorescence channel images were taken at a resolution of 0.61 µm per pixel every 45 min for a total of 315 min. Images were exported as 16-bit grayscale files and analyzed using CellProfiler^{37 (link)} to quantify cells and phagocytized particles. CellProfiler pipeline description are included in Supplementary Materials.