In vitro migration assay protocol

In vitro migration assays were performed using uncoated 24-well chambers/microfilters (BD), as previously described^{4 (link)–7 (link)},⁸⁹. Briefly, after rehydration of the chambers, cells (2.5 × 10⁴ cells per chamber) in 500 µL phenol red-free DMEM/F12 without FBS (Life Technologies) were seeded onto the upper chamber. In the lower chamber, 750 µL phenol red-free DMEM/F12 plus 10% charcoal-stripped FBS (Life Technologies) were added. IL-1β and/or TNFα at the indicated concentrations or vehicle only was then added into the upper chamber. Cell motility/migration was measured as the number of cells that migrated from a defined area of the uncoated microfilter through micropores in 48 h. The micropore filters were stained with 0.5% crystal violet, and the number of cells that migrated through filters was counted in the entire area of each filter. To count cell numbers objectively, a computerized image analysis system consisting of a light microscope (Leica, Lyon, France) (X20 objective, X10 ocular) and a color charge-coupling device camera (Sony, Paris, France) were utilized. All experiments were performed in duplicate.