Licon master. The agarose gel membrane was then placed on a 1 inch61 inch glass slide and was sandwiched between a Plexiglas manifold and a stainless metal frame in a biohood. The operation principles are shown in theRoles of Two Cytokines in Tumor Cell MigrationFigure 1. Microfluidic device setup and data acquisition. A. An image of the microfluidic device on the microscope stage. A penny is placed on the side for scale. B. Schematic illustration of the microfluidic device. Three parallel channels are patterned on a 1-mm thick agarose gel membrane. A stable linear gradient 10457188 is generated across the center channel by flowing solutions of chemokine and buffer through the source and the sink channels respectively. A mixture of cells (1 million cells/ml) embedded in type I collagen (1.5 mg/ml) is seeded in the center channel. All three channels are 400 mm wide and 250 mm deep, and the ridges between the channels are 250 mm wide. C. 3D reconstruction of a z-stack of 65 images (5 mm each) of the cellembedded collagen matrix viewed from x-y plane (top view) and the x-z plane (side view); scale bar, 50 mm. D. Cell trajectory plots (60 cells each) from the four conditions indicated. In the last panel, the uniform 0.25 nM EGF is generated by supplying 0.25 nM EGF solutions along the two side channels. Each colored line represents one cell trajectory tracked in 16 h. doi:10.1371/journal.pone.0068422.gby a temperature controlled chamber (Weather Station, Precision Control LLC) set at 37uC. For each experiment, we typically imaged 8 positions (2 on each device, with 4 devices on one chip) using the x-y controlled stage (OptiScan II, Prior Scientific, Inc., Rockland, MA). The images were captured every 5 minutes for 16 hours using the bright field microscopy (206objective, Olympus IX81, Center Valley, PA), an image acquisition software SlideBook (Intelligent Imaging Innovations, Inc., Denver, CO) and a CCD camera (Orca-ER, Hamamatsu, Bridgewater, NJ). For the data reported here, we imaged one plane close to the center of the channel in the vertical direction. These experiments were repeated at least once. Cell trajectories were obtained first using a Manual Tracker in ImageJ (Nea was cut, and the graft was placed on the host National Institutes of Health) from the time series images as shown in Figure 1D. Cell speed U’ (H the number of leukocytes in the NE and MC was length of the trajectories divided by time), the velocity Vx ‘ (displacement along the gradient direction divided by time), cell persistence length P’ (the displacement of a cell trajectory divided by the length of the trajectory), and the cell persistence length along gradient direction Px ‘ (the displacement of a cell trajectory along the gradient direction divided by the length of the trajectory) were then computed from the cell trajectories of 16 hour duration (See Figure 2E) using an in house Matlab program (The MathWorks, Inc., Natick, MA). Here, only motile cells (U’ .0.2 mm/min) were included for further data analysis, which usually accounted for ,50 of the total cell population. The average speed, velocity and persistence length were computed from about 120 or more cells under the same chemical gradient, which usually come from at least two separate experiments. To minimize the experiment-toexperiment variation, speed U is normalized by the average speed of the control cells (no chemical gradients), and velocity along the direction of gradient, Vx , is computed as the average velocity along x-direction subtracted by that of the control group, and divided by the average speed.Licon master. The agarose gel membrane was then placed on a 1 inch61 inch glass slide and was sandwiched between a Plexiglas manifold and a stainless metal frame in a biohood. The operation principles are shown in theRoles of Two Cytokines in Tumor Cell MigrationFigure 1. Microfluidic device setup and data acquisition. A. An image of the microfluidic device on the microscope stage. A penny is placed on the side for scale. B. Schematic illustration of the microfluidic device. Three parallel channels are patterned on a 1-mm thick agarose gel membrane. A stable linear gradient 10457188 is generated across the center channel by flowing solutions of chemokine and buffer through the source and the sink channels respectively. A mixture of cells (1 million cells/ml) embedded in type I collagen (1.5 mg/ml) is seeded in the center channel. All three channels are 400 mm wide and 250 mm deep, and the ridges between the channels are 250 mm wide. C. 3D reconstruction of a z-stack of 65 images (5 mm each) of the cellembedded collagen matrix viewed from x-y plane (top view) and the x-z plane (side view); scale bar, 50 mm. D. Cell trajectory plots (60 cells each) from the four conditions indicated. In the last panel, the uniform 0.25 nM EGF is generated by supplying 0.25 nM EGF solutions along the two side channels. Each colored line represents one cell trajectory tracked in 16 h. doi:10.1371/journal.pone.0068422.gby a temperature controlled chamber (Weather Station, Precision Control LLC) set at 37uC. For each experiment, we typically imaged 8 positions (2 on each device, with 4 devices on one chip) using the x-y controlled stage (OptiScan II, Prior Scientific, Inc., Rockland, MA). The images were captured every 5 minutes for 16 hours using the bright field microscopy (206objective, Olympus IX81, Center Valley, PA), an image acquisition software SlideBook (Intelligent Imaging Innovations, Inc., Denver, CO) and a CCD camera (Orca-ER, Hamamatsu, Bridgewater, NJ). For the data reported here, we imaged one plane close to the center of the channel in the vertical direction. These experiments were repeated at least once. Cell trajectories were obtained first using a Manual Tracker in ImageJ (National Institutes of Health) from the time series images as shown in Figure 1D. Cell speed U’ (length of the trajectories divided by time), the velocity Vx ‘ (displacement along the gradient direction divided by time), cell persistence length P’ (the displacement of a cell trajectory divided by the length of the trajectory), and the cell persistence length along gradient direction Px ‘ (the displacement of a cell trajectory along the gradient direction divided by the length of the trajectory) were then computed from the cell trajectories of 16 hour duration (See Figure 2E) using an in house Matlab program (The MathWorks, Inc., Natick, MA). Here, only motile cells (U’ .0.2 mm/min) were included for further data analysis, which usually accounted for ,50 of the total cell population. The average speed, velocity and persistence length were computed from about 120 or more cells under the same chemical gradient, which usually come from at least two separate experiments. To minimize the experiment-toexperiment variation, speed U is normalized by the average speed of the control cells (no chemical gradients), and velocity along the direction of gradient, Vx , is computed as the average velocity along x-direction subtracted by that of the control group, and divided by the average speed.