Boyden chamber migration assay

HUVECs were serum-starved (EBM-2 + 0.5% FBS) overnight. Cells (50,000 per well; 24-well format, BD Falcon) were seeded in triplicate on collagen-coated inserts of a transwell chamber with 8-μm-sized pores (BD Falcon) and incubated in serum-free medium (EBM-2, Lonza) for 3 hours. After starvation, 1.2 ml of serum-free medium supplemented with stimulant [hVEGF-A (100 ng/ml) or S1P (1 μM)] was placed in the lower transwell chamber. Cells were allowed to migrate for 6 hours toward the lower chamber stimulant, followed by fixation of the cells with 4% PFA for 15 min and incubation with 0.1% crystal violet for 10 min to stain the cells. Cells remaining on top of the membrane insert were cleaned off with a cotton swab, and the migrated cells at the bottom of the insert were imaged at ×10 magnification. Cell migration was measured by counting the number of migrated cells per field, five fields for each well, and three wells of each condition were averaged for quantification.