Live cell wound migration

Live cell imaging experiments have been performed as previously described [35]. Briefly, 50,000 cells were seeded in half-area 96-well plates with a glass bottom (Corning 4580 high content imaging plate). Cells were allowed to grow under normal cell culture conditions for 48 h, including medium refreshment after 24 h. For CtsK inhibition MK-0822 was added to the medium for 24 h before migration start. At start of migration, wounds were generated, the cells carefully washed with pre-warmed migration medium (RPMI 1640, 1 mg/ mL bovine serum albumin (BSA) and 0.1 mg/ mL gentamicin) and migration medium added, respectively containing MK-0822. Wound migration was followed for 30 h by collecting an image series of 121 times points at 4X magnification in a NikonA1+ confocal laser microscope system equipped with temperature- and CO₂-regulation. Migration distances were calculated by image processing and calculations performed with use of the NIS-Elements Advanced imaging software 4.3.0 (Nikon, Tokyo, Japan). Migration velocity was assessed by linear regression analysis in GraphPad Prism 6.