2 well culture insert 24 well plates

BMDMs were seeded at a density of 50,000 cells/insert in 2-well culture insert 24-well plates (Ibidi). Cells were incubated at 37°C in 5% CO₂ for 24 h. Culture inserts were then carefully removed followed leaving an unpopulated “scratched” area and the cell monolayer with fresh complete medium and imaging of the scratch area using an EVOS digital inverted light microscope. Extent of microglia cell migration into the scratch area was quantified using a customized ImageJ script.
iMGL cultured for 30 days were replated in a clear bottom 96-well plate (ImageLock) coated with poly-D-lysine (1 mg/mL) at 40,000 cells per well and were allowed to rest at 37°C in 5% CO₂ for 1 h. Cells were then mechanically removed from the center of the well with a Incucyte^® 96-Well Woundmaker Tool. Migration of iMGL to the generated wound was measured in images acquired in Incucyte S3 (Sartorius) using the Relative Wound Density metric (percentage of cells that migrated to the wound compared to the cells that remained in the unscratched area).

Primary microglia were seeded at a density of 30,000 cells/insert in 2-well culture insert 24-well plates (Ibidi). Cells were incubated at 37 °C in 5% CO₂ until reaching approximately 80% confluence. Culture-inserts were then carefully removed followed by washing of the cell monolayer with fresh complete medium and imaging of the scratch area using an EVOS digital inverted light microscope. Primary microglia were treated with 1 μM of CC-292 for 24 h and the scratch area re-imaged. The extent of microglia cell migration into the scratch area was quantified using ImageJ.