Two-month-old CX3CR1+/GFP mice were subjected to PLX5622-mediated microglial depletion for 7 days and allowed to undergo microglial repopulation for 2 months. Retinal explants were prepared from these animals and from age-matched CX3CR1+/GFP mice that had not been subjected to microglia depletion, as previously described (23 (link)). Baseline dynamic behavior of repopulated microglia versus endogenous microglia was monitored and recorded using time-lapse confocal microscopy; responses to the addition of exogenous ATP (5 mM in Ringer’s solution, Sigma-Aldrich) into the imaging chamber were also recorded. Time-lapse image stacks were captured every 24 s for up to 30 min. Two-dimensional time-lapse movies were created from maximum intensity projections in the z dimension and aligned in the x-y plane using the StackReg plugin of NIH ImageJ software. Rates of process extension and retraction of individual microglial cells were measured as previously described (24 (link)).