Movie 2

3D chemotaxis live imaging of cells migrating with a shallow gradient of S1P and a low concentration of 12-S-HETE. The gradient is higher to the left. Cell responding to the gradient of S1P and HETE are more polarized and migrate greater distances. The cells turn frequently, yet overall the direction of migration is to the left. Blood was isolated from Botryllus colonies, diluted ½ with filtered seawater, filtered through 10μm cell strainers and mixed with 50% Matrigel. To achieve a shallow gradient of S1P and 12-S-HETE, 6μl of 0.2 μM S1P and 6μl of 50nM 12-S-HETE were added to the corner of the left reservoir containing 60μl of filtered seawater. Live imaging was performed on a Leica SP8 confocal microscope at 15s intervals over a time period of 45 minutes. Every particle visible in these movies is a cell, even though many are not within the focal plane.