Insert the cell culture plate in the microscope holder, and set chamber parameters: T 37°C and CO2 5%.

Using X20 magnification chose 20 random fields;

Set exposition of the fluorescent channel to have a sharp image of microglia bodies and branches;

Records the live fluorescent microglia for 2 h taking a picture every 5 min.

Save the recorded file as a “.zvi”.

Open ".zvi" file with Fiji software as hyperstack and tick the “split channel” option;

close the bright field channel and start to elaborate the fluorescent channel (microglia);

subtract the background using “Process › Subtract Background”(identifier: legacy:ij.plugin.filter.BackgroundSubtracter);

defined threshold (foreground) that corresponds to green fluorescent objects using “Image › Adjust › Threshold” (identifier: legacy:ij.plugin.frame.ThresholdAdjuster). Keep the threshold consistent between acquisitions;

apply despeckle function “Process › Noise › Despeckle” (identifier:legacy:ij.plugin.filter.RankFilters("despeckle"));

apply smoothing function “Process › Smooth” (identifier: legacy:ij.plugin.filter.Filters("smooth"));

set the measurement: “Analyze › Set Measurements” (identifier: legacy:ij.plugin.filter.Analyzer("set")), and tick “Area”, “Center of Mass”, “Feret’s Diameter” and “Shape Descriptors”;

for each microglia select the area that contains the microglia in each time-frames using “Edit › Options › Roi Defaults” (identifier:legacy:ij.gui.RoiDefaultsDialog);

run analyze particles “Analyze › Analyze Particles” (identifier:legacy:ij.plugin.filter.ParticleAnalyzer), set size (micron^2):130-infinity, circularity: 0.00-1.00; tick “display results”;

process “all images”;

copy the data in an Excell file;

repeat the steps from 13 to 16 for each microglia.

Among the "Shape Descriptors", keep the values of “Area”, “Solidity”, “FeretAngle”, “XM” and “YM”;

to calculate the distance covered by the cell during the time-lapse use the coordinates of the center of mass and sum the distance covered in each time frame assuming that the distance between frames corresponds to the cathetus of a right triangle made by X-axis and Y-axis displacement;

to calculate the rotation sum the “FeretAngle” taking into account that it is the angle among Ferret’s diameter and parallel line to the cell contour only on x-axis;

calculate the median and the CV% of "Solidity" and "Area";

to perform the cluster analysis use each parameter obtained from the analysis; use the values of the vehicle and treated cells and identify the median parameter for the experiment;

use the identified median as a threshold to cluster the cells in two groups (over or under the median);

combine two parameters to generate four different clusters.