Although understanding the collective migration of cells such as that seen in epithelial sheets is essential for understanding diseases such as metastatic cancer this motion is not yet as well characterized as individual cell migration. the motion of the cells into affine and non-affine components using the metric adds spatial information about which regions are more or less chaotic. This allows us to assess whether the chaotic features of the flow field are correlated Methscopolamine bromide with a cell’s distance from the leading migration edge or other spatially localized features. The tracks give information about individual cells; by combining PIV data with individual cell tracks we can consider the motion and flow of the migrating epithelial sheet. To parameterize an individual cell’s deviation from Methscopolamine bromide the motion predicted by the average flow field we use the previously defined parameter moves in a distinct manner compared to what is predicted by the smooth local flow field. Regions in the sheet with high can be considered regions of high rearrangement. To illustrate the use of these two metrics we analyze a migrating two-dimensional culture of epithelial cells. We find that statistics such as average speed vary from experiment to experiment even under the same conditions. However by considering our metrics for rearrangement and stretching we are able to quantify trends that hold across different experiments. We find that despite the radial migration of our monolayer most of the stretching of the sheet is limited to a small region near the leading migration edge. We also find that rearrangements within our cell sheet increase as the local cell number density increases a surprising trend that could be used to isolate active properties of the migrating cells. These results suggest that and FTLE data will be useful in distinguishing and classifying how cells migrate collectively in many different systems under varying conditions making them a promising tool for future cancer studies. 2 Materials and Methods 2.1 Cell Culture and Microscopy MCF10A cells were cultured in DMEM/F12 media supplemented with 5% horse serum 10 μg/ml insulin (Invitrogen) 10 ng/ml EGF (Peprotech Rocky Hill NJ) 0.5 μg/ml hydrocortisone and 100 ng/ml cholera toxin (both Sigma St. Louis MO). The cells were kept in a humidified atmosphere at 37 °C and 5% CO2. For the migration assay cells were plated in 12 well glass bottomed plates coated with collagen IV (10μg/ml for approximately three days at 4 °C) and allowed to culture overnight in DMEM/F12 containing 1% horse serum (unless otherwise noted to be 5% horse serum). Figure 1 provides an illustration of Methscopolamine bromide the plating technique. Cells were then imaged for 24 hours on an incubator microscope kept at 37 °C and 5% CO2 (Zeiss Observer.Z1 Zeiss Goettingen Germany). Phase contrast images of the cells were acquired at 2 minute intervals using a 10× objective. Figure 1 (a) Cartoon of migration assay. Cells were plated in a 2 μl drop and allowed to adhere the collagen surface before the well was filled with media. (b c) Phase contrast image of leading edge of cell migration (scale bar 50 μm). Rabbit Polyclonal to ZNF287. (d e) Velocity … 2.2 Image Analysis Particle Image Velocimetry (PIV) analysis is performed using the MatPIV MATLAB toolbox (J. Kristian Sveen: http://folk.uio.no/jks/matpiv/ GNU general public license). We use multiple iterations of interrogation window sizes starting with two iterations of 64 × 64 pixel windows (41.6 μm × 41.6 μm) and finishing with two iterations of 32 × 32 pixel windows (20.8 μm × 20.8 μm). At each interrogation 50 overlap is used and the windows are cross-correlated. After the final iteration outliers Methscopolamine bromide are detected using a signal-to-noise filter. Vectors with a signal-to-noise ratio less than 1.3 are replaced by linearly interpolated values. In the case of movies involving a leading edge of the cell Methscopolamine bromide sheet the edge of the sheet is segmented and the detected edge is used Methscopolamine bromide to trim the PIV field to remove the zero velocity contribution of the empty space from any further metrics. We track individual nucleoli using an algorithm adapted from hydrodynamic turbulence studies implemented in Matlab and available online [33]. We begin by calculating each data set’s mean image which we take as the.