As mdw live cell imaging system

Cells were grown to confluence and after 24 hours were scratched with a pipette tip before being washed once in appropriate serum containing medium and placed into fresh serum containing medium. Cells were treated with MEK inhibitors or DMSO at 1μM and cRGDfV at 2.5 μM as appropriate. Images were taken every 10 minutes for >84 time points (15 hours) for 6 different areas per condition. Cells were imaged using an AS MDW live cell imaging system (Leica) using a 20x/NA 0.50 Plan Fluotar Ph2 objective in brightfield. Micromanager imaging software and point visiting mode were used to allow multiple positions to be imaged within the same timecourse while cells were maintained at 37°C and 5% CO2. Images were collected using a Cascade II EM CCD camera (Photometrics). At least 5 cells per time-lapse position (giving 30 cells tracked per condition) were individually manually tracked using the ImageJ plugin MTrackJ [69 ] for the nucleus position every 3 frames (i.e. using 30 minute timepoint intervals). Cells were unbiasedly chosen and in general either started or ended at the leading edge of the scratch wound. The Chemotaxis and Migration Tool [70 ] was used to calculate the average speed and directional persistence for each given condition, where persistence is the ratio (displacement from initial position)/(total distance travelled).

Cells were cultured in 24-well tissue culture plates, and treated with 1 μM Taxol, 5 μM ABT-737, or 10 μM 4-OHT as detailed in text, and imaged at 15 min intervals for 50 h, maintained at 37 °C and 5% CO₂. Images were acquired on an AS MDW live cell imaging system (Leica) in brightfield using a 20× HC Plan Fluotar objective, using point visiting to allow multiple positions to be captured within the same time course. Image stacks were analysed using ImageJ, single cells identified and followed manually to determine fate.

In all, 1 µM Taxol (Sigma-Aldrich), 5 µM ABT737 (Sigma-Aldrich) or DMSO diluted in complete growth media was added to cells 30 min prior to imaging. Images were acquired at 15-min intervals for 48–60 h on an AS MDW live-cell imaging system (Leica) in bright field using a ×20 objective and utilising imaging software Image Pro 6.3 (Media Cybernetics Ltd). Point visiting was used to allow multiple positions to be imaged within the same time course, and cells were maintained at 37 °C and 5% CO₂. Image stacks were analysed in ImageJ (National Institutes of Health), where single cells were identified and followed manually using cellular morphology to determine cell-cycle progression and fate. Cells were described as being in mitosis from the initial point at which a cell began to round, until the first frame of cytokinesis or membrane blebbing. Cells that exhibited morphology associated with apoptosis, such as membrane blebbing, shrinking of the cell body, and in the later stages, the formation of apoptotic bodies or secondary necrosis was described as having undergone apoptosis in mitosis. Cells that exited mitosis as a single cell, or as two or more non-identical daughter cells, were grouped together under the definition of having undergone mitotic slippage.

Activated HSCs were cultured in 24-well plates at a density of 5,000 cells per well. Single-cell tracks were acquired using live-cell imaging at 10 min intervals over a 24 h period (AS MDW live cell imaging system, Leica). Multiple position imaging was gathered using Image Pro 6.3 (Media Cybernetics Ltd) and a Coolsnap HQ (Photometrics) camera with a Z optical spacing of 0.2 μm. Cell movement over the 24 h period was analysed using ImageJ software with the MTrackJ plug-in. Total length of the cell track (in μm) was generated for multiple cells (details are provided in the figure legends), averaged and normalized to control. Where individual cell migration tracks are shown, the co-ordinates at 10 min intervals were plotted relative to each cell’s starting point (set at 0,0).