Random migration assay
Microglia were seeded at a density of 12,000 cells/well in a PDL coated
96-well ImageLock Plate (Essen Bioscience, 4379) and stained with
CellTracker Red CMTPX Fluorescent Probe (4.5 µM) (Life Technologies).
DMSO (0.2%), LPS (10 ng/ml), and CdA in different concentrations alone
(0.1-10 µM) or together with LPS were added, and cells were placed in
the IncuCyte Zoom live cell imaging system (Essen BioScience, USA).
Pictures were taken every 20 min for 24 hours. Single cell motility was
quantified using the Fiji plugin TrackMate for semi-automated particle
tracking (21). To detect individual cells in TrackMate, the Laplacian of
Gaussian (LoG) detector with estimate spot diameter of 24.12 μm and a
threshold of 0.3 was used. The simple linear assignment problem (LAP)
tracker with a linking maximum distance of 60 µm, a gap-closing max
distance of 15 µm and a gap closing max distance of 2 was used to track
cell migration through the time course movies. The number of spots in
track was set to 71.09 to exclude cells, which were not detectable
throughout the movies from the analysis.