2.13. Scratch wound assay

Microglia conditioned medium (MGCM) was collected from the N9 microglial cell line after culturing the cells for 24 hours in DMEM, 10% FBS, 1% penicillin‐streptomycin, and kept at 5% CO₂ and 37°C. The media collected were either the control media (MGCM) or media generated after different treatments of the cells, either with 200 ng/mL LPS (MGCM + LPS) or with both LPS and 5 μM PFT‐μ (MGCM + LPS + PFT‐μ). LPS treatment of N9 cells results in a robust pro‐inflammatory response, analogous to cultures of primary microglia,²⁸ and the use of this cell line allowed for the collection of larger volumes of media to be used for experiments. Cells were preincubated for 2 hours with PFTμ prior to the addition of LPS. Conditioned media were collected, spun down to remove dead cells and debris, and stored at −80°C.

Reference lines were etched horizontally into the bottom of 6 well plates using a razor. Scratches were then made, perpendicularly, within the confluent mixed cortical cultures (MCC). Three horizontal etches and three vertical scratch wounds made a total of 9 intersecting regions of interest within each well. After scratch induction, MCCs were treated with either control media, 5 μM PFTμ, MGCM, MGCM + LPS, MGCM + LPS + PFTu, or MGCM + LPS with the addition of 5 μM PFTμ to the MCC. After 48 hours of incubation, cells were fixed in 4% PFA, blocked with 3% NGS/PBST, and immunostained with GFAP (Cell Signaling, 1:1000) and Texas Red‐X Phalloidin (Invitrogen #T7471). Aqueous DAPI was added to the wells for 10 minutes and the wells were then washed with PBS. Well plates were imaged using a Zeiss Axiovert 200 M inverted microscope. One‐way ANOVA was used to compare treatment groups. Nine images were taken per experiment, which was independently replicated three times.