0 hours incubation, cells were fixed and stained with 00% methanol, 0% crystal violet and cotton swabs were used to remove the cells from the upper side of the membrane. An Axio Observer.A0 microscope (Carl Zeiss, Jena, Germany) was used to record 0 micrographs per well. Photoshop CS0 (Adobe, San Jose, CA) and a proprietary script were used for automated quantification of migration.  