Quantifying Cell Migration Using Boyden Chamber Assay

Cell migration was assessed using a 48-well Boyden microchemotaxis chamber (Neuro Probe Inc., Gaithersburg, MD, USA), as in a previous report [30 (link)]. Briefly, lower chambers were loaded with DMEM containing 0.1% BSA and different concentrations of SKAFAb or rhEGF (5 ng/mL). A membrane coated with type Ι collagen was then laid over medium in lower chambers. Upper chambers were loaded with cells (5 × 10⁴ cells/well) in DMEM containing 0.1% BSA. Chambers were then incubated for 210 min at 37 °C. Membranes were removed and then fixed and stained using Diff-Quick (Baxter Healthcare, Miami, FL, USA). The lower part images of the membrane were captured in three randomly selected regions of each well using an optical microscope (×200). The number of migrated cells in captured images of each well was counted and the mean values were calculated. Each experiment was repeated four times. Migration level was expressed as a percentage of control (the untreated state).