Movie prepared from time series light microscopy images illustrating a wound healing assay. A monolayer of MDCK (Madin-Darby Canine Kidney) epithelial cells is scratched to create a 'wound' about 300 micrometers in width, after which the growth of the cells to fill the wound is monitored by recording images over the course of 15h. This movie is part of a group of 12 that includes control cells (CIL:44501, 502, 506, 507, 508) and after addition of Hepatocyte Growth Factor /Scatter Factor (HGF/SF) to activate HGF/SF-Met signaling (CIL:44503, 504, 505, 509, 510). Also in the group are two movies, one control (CIL:44511 created from CIL:44501), and one HGF/SF treated (this image, created from CIL:44512). For additional wound healing assays see CIL:43401 and images grouped with it.
Madin-Darby Canine Kidney (MDCK) epithelial cells expressing YFP-membrane were maintained in DMEM supplemented with 5% fetal FCS in a 37°C, 5% CO2 incubator. Wound healing assay: Cells were grown to 90% confluence in 24-well plates. Prior to scratching, the cells were starved by changing the medium to DMEM plus 0.1% FCS (starvation medium) for 24 hours. The medium was then changed to either fresh starvation medium (control), or starvation medium with 80 ng ml-1 HGF/SF for an additional 2 hours. A scratch of approximately 300 μm in width was generated using a 200 μl tip. The plate was subjected to time lapse microscopy in a stage incubator (OKOLAB, Italy) on a computer-controlled motorized stage of a confocal microscope (CLSM-510, Carl Zeiss, Germany), used in non-confocal DIC mode, with a 10x (NA 0.30) objective. Image acquisition was initiated 2 hours post scratching. Images were acquired every 15.7 minutes for 15 hours. The coordinates of each scratch were predefined, and a macro that repetitively positions the field of view at each point was executed. The acquired differential interference contrast (DIC) channel of the time-lapse sequence was used for the analysis.
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