Treat passage two AdMSCs with media containing 10 nanograms per milliliter of TNF alpha for 24 hours or without TNF alpha for unstimulated cells. After washing and incubating the cells in serum-free media for four hours, add 25, 000 AdMSCs to each insert and incubate for 24 hours at 37 degrees Celsius. Aspirate the contents of both well and insert, then wash twice with PBS leaving one milliliter of PBS in each well.
Using forceps, remove the inserts one by one and gently but thoroughly wipe the top chamber with a cotton swab to remove unmigrated cells. Aspirate the remaining PBS and pipette 500 microliters of crystal violet solution to the well and the top chamber of the insert. Aspirate the crystal violet solution from the well and top chamber of the insert after one hour incubation.
After washing and wiping the top chamber, place the insert in a new 24 well plate containing one milliliter of PBS. Using an inverted microscope with a camera, image four random areas towards the insert's center under a 10 times objective. Upload the images to the preferred image processing software and adjust the background for enhanced contrast with purple stained cells.
CLIA infected with 22L showed increased mRNA expression for inflammatory markers CCL2, CCL5, and IL1 beta, and the astrocyte marker S100 beta compared to those treated with NBH. Coculturing with AdMSCs decreased expression of the inflammatory cytokines CCL2, CCL5, and IL1 beta, but not TNF alpha for both MBH and 22L infected cells. BV2s cocultured with AdMSCs showed a decrease in mRNA for the inflammatory markers IL1 beta, IL-6, TNF alpha, and the complement protein C1qa in both NBH and RML treated cells.
Additionally, AdMSCs decreased the M1 microglia gene CD16 and increased the M2 microglia marker ARG-1. The in vitro migration assay suggested that AdMSCs showed significant migration toward media containing 1%RML.
