Video microscopy must be used when performing an ibidi chemotaxis and migration assay. Without video microscopy, there is no access to, or analysis of, the chemotaxis effects.
3) Cell Tracking
Cell tracking is the only way to access the quantification of cell movement between the frames of a temporal stack. The tracking can be done manually, or you can do the tracking automatically by using special software. Automated tracking algorithms need distinct objects, such as fluorescent labeled cells. After tracking the cells' traces, their (x, y) values are available for each point of time (t).
Cells’ traces are visualized after tracking with ImageJ plugin “Manual Tracking”.
Data table with (x, y) positions for each point of time (t).
4) Plotting the Data
After the coordinate transformation, the ibidi Chemotaxis and Migration Tool automatically sets all initial points to (0,0). Cell trajectory plots can be easily created. For better visualization of chemotaxis effects, additional information can be added to the plots (e.g., color information of cells moving up/down).
The simplest way to interpret data is to make a visual inspection of the cell trajectories (plots). Strong and significant chemotaxis effects towards one specific direction can easily be seen. Furthermore, significant differences in cell velocity and directionality between the chemotaxis and the control experiment can be distinguished. When dealing with strong chemotaxis effects, as seen in our example, or with total chemotaxis inhibition, simple interpretation might be sufficient. We also recommend taking into account the provided Rayleigh test. In our example, you can also see that the total amount of chemoattractant, without any gradient, leads to a different migration behavior of the cells.
In addition to the optical impression of the plots, profound parameters can be used to prove a chemotaxis effect or a hypothesis. Parameters, such as the displacement of the center of mass (M) or the forward migration indices, are valid measures when they are compared to the right control experiments. In our experiment example, the yFMI of the (+/-) measurement is significantly higher than the xFMI and it is also higher than the xFMI and yFMI of the control experiments (-/-) and (+/+).