Scratch Wound Assay

To analyze the migratory capacity of astrocytes undergoing different treatments, the scratching astrocyte monolayer method was used to examine the astrocyte migratory ability[32]. Briefly, 500,000 astrocytes were transferred into the wells of a 6-well plate and cultured for at least 24h until cell confluence reached greater than 99%. The confluent astrocyte monolayer was scratched in a straight line with a sterile pipette tip (200 µL) following the marker guide sheet under the 6-well plates. The cells were washed 3 times with PBS to remove the detached cells and debris. Concomitantly, the astrocytes were treated with 1% FBS-DF12 containing IL-1α (3 ng/mL) + TNFα (30 ng/mL) + C1q (400 ng/mL), Y27632 (10 µM), and IL-1α (3 ng/mL) + TNFα (30 ng/mL) + C1q (400 ng/mL) + Y27632 (10 µM). Next, bright-field images (magnification 10×) of cells were captured at 0, 6, 12, and 24h after the scratches were made. The ratio of cell migration was calculated as the percentage of the remaining cell-free area against the initial scratch area. At a 10×scaling, a contour was drawn around the cells at the edge of the scratch and the wound area was calculated in square microns.