Macrophage and Smooth Muscle Cell Migration Assays

Macrophage migration was measured using a modified Boyden chamber method54 (link). Macrophages from WD-fed ApoE^−/− and ApoE^−/−:Pak1^−/− mice were suspended in DMEM medium and plated on Matrigel-coated 8-μm cell culture inserts at 5 × 10⁴ cells per insert. MCP-1 was added to a final concentration of 50 ng ml⁻¹ to the lower chamber and the cells were incubated for 8 h at 37 °C. The inserts were then lifted, non-migrated cells on the upper surface of the membrane were removed with a cotton swab and the membrane was then fixed in methanol and stained with DAPI (4′,6-diamidino-2-phenylindole). The DAPI-positive cells on the lower surface of the membrane were counted under an inverted microscope (Carl Zeiss AxioVision AX10) and the cell migration was expressed as the number of cells per field of view. SMC migration was measured by wound-healing assay. Briefly, SMCs were plated at 2 × 10⁵ cells per ml in each chamber of the ibidi culture inserts, grown to full confluency and growth arrested. Following a 24-h growth-arresting period, the inserts were removed using sterile tweezers and 2 ml of DMEM containing 5 mM hydroxyurea was added to the culture dish, and incubation continued for 48 h. The migrated cells were observed under Nikon Eclipse TS100 microscope with × 10/0.25 magnification and the images were captured with a Nikon Digital Sight DS-L1 camera.