Microglial Cell Migration Assay

Cell migration assay was performed in a 48-well microchemotaxis chamber (Boyden Chamber, Neuro Probe, Gaithersburg, MD, USA) as we published [39 (link)]. Briefly, N9 cells were resuspended in serum-free RPMI and 50 μL of cell suspension was placed into each top well (2–4 × 10⁴ cells per well). The bottom wells were filled with serum-free RPMI (basal medium) alone, or with ATP (10 µM), and LPS (300 ng/mL) diluted in basal medium. Microglial cells were allowed to migrate for 6 h through a polycarbonate track-etch membrane with polyvinylpyrrolidone (PVP) (Neuro Probe) towards the solution in the bottom wells. Afterwards, the membrane was removed and the bottom side fixed with cold methanol. Cells were stained with 10% Giemsa in PBS (w/v), freshly prepared and filtered. The number of total cells was counted in ten microscopic fields with ImageJ software (original magnification: 100x) acquired to observe the complete well using Leica IM50 software and Leica DFC490 camera (Leica Microsystems, Wetzlar, Germany), adapted to an AxioSkope HBO50 microscope (Zeiss). For each experiment, at least three wells per condition were acquired.