Wskm stage top incubator

Scratch wound assays were performed in a 24-well plate. Media was introduced to the well, followed by the seeding of ~ 10,000 cells. The cells were cultured for at least 3 days beyond the attainment of confluency (usually 7 days total), then scratched with a P200 pipette tip down the middle. Live imaging of the “wound” region was performed for 24, 36, or 72 hours (at four frames per hour) on the Tokai Hit WSKM stage top incubator (Tokai Hit CO.,Ltd., Shizuoka, Japan). Since the positions of the imaging regions were memorized on the motorized stage, it is possible to obtain the image before and after the scratch on the same locations.

All substrates were coated via incubation with 1:100 dilution of Geltrex® (0.15 mg/mL concentration) for 30 minutes, then pipetted off and let dry for another 30 minutes at 37 °C. The Geltrex provides a good mimic of a basement membrane, and contain laminin, collagen IV, entactin, and heparin sulfate proteoglycan, but no fibronectin. The 1:100 dilution is recommended by the manufacturer to prevent the formation of a thick gel on the substrate. We have shown through scanning electron microscopy (SEM) and fluid permeability study that the Geltrex coating performed this way did not occlude the pores. Media was introduced to the bottom gasket, followed by the seeding of ~160 cells onto the 2 mm x 2 mm substrate area to minimize the frequency of cell collision, which may complicates the migration analysis. Cells were allowed to adhere to the coated substrates for three hours prior to imaging. 24-hour live imaging (at four frames per hour) was performed on the Tokai Hit WSKM stage top incubator (Tokai Hit CO.,Ltd, Shizuoka, Japan).