Use a p200 pipet tip to create a scratch of the cell monolayer. Wash the plate once and replace with the desired medium. If time-lapse microscopy is used, CO2-independent media (e.g., HEPES-buffered media) may be required. The time-lapse microscope is used for acquiring the images from the same field automatically. It can be equipped with a stage incubator either with only temperature control or with both temperature and CO2 control. For the chamber with only temperature control, it is necessary to use CO2-independent medium during the assay. Nonetheless, it is not very practical to examine cell migration over a period of over 18 h by using the CO2-independent medium.