Following the sterile water rinse, devices that were destined for cell migration studies were filled with 0.2 mg/mL fibronectin (Millipore) in phosphate buffered saline (PBS) and incubated for at least 2 hours at 37 °C. Cells were trypsinized, counted and suspended in complete medium (DMEM containing 10% fetal bovine serum) at a density of 5 × 106 cells/mL. The migration devices were rinsed with complete medium and aliquots of 4 μL were added to the cell inlet ports, corresponding to 20,000 cells. The cells were then allowed to attach overnight before changing the medium.
Migration studies to assess the effect of the chemoattractant were carried out in the incubator. Twenty four hours after seeding NIH 3T3 mouse embryonic fibroblasts (MEF), images were taken of the migration devices. The medium was then changed in both reservoirs simultaneously: the medium on the side of the cells was replaced with complete medium, whereas the reservoir towards which the cells migrated was replaced with complete medium containing concentrations of PDGF varying from 0 to 200 ng/mL. Migration devices destined for live cell imaging were treated similarly: the medium was replaced 24 hours after seeding with phenol-red free medium containing 25 mM HEPES (Gibco). Immediately following medium change, the devices were covered with a coverslip seal the device and limit medium evaporation.
Migration studies to assess the effect of the chemoattractant were carried out in the incubator. Twenty four hours after seeding NIH 3T3 mouse embryonic fibroblasts (MEF), images were taken of the migration devices. The medium was then changed in both reservoirs simultaneously: the medium on the side of the cells was replaced with complete medium, whereas the reservoir towards which the cells migrated was replaced with complete medium containing concentrations of PDGF varying from 0 to 200 ng/mL. Migration devices destined for live cell imaging were treated similarly: the medium was replaced 24 hours after seeding with phenol-red free medium containing 25 mM HEPES (Gibco). Immediately following medium change, the devices were covered with a coverslip seal the device and limit medium evaporation.
