Chemotaxis

Concentration gradients of a large variety of substances induce a directed motion of cells (chemo-taxis). Due to its importance for angiogenesis, oncology, neurology, and especially immunology, the question of migration under a special stimulation gains a lot of interest. Until now, there was no simple system to study these complicated correlations in easy optical assays.

ibidi chemotaxis slides

To overcome the disadvantages of existing chemotaxis assays, the ibidi research team developed µ-Slides for chemotactical analysis. The special chambers allow high resolution microscopy, convenient liquid handling, and live cell imaging under defined linear concentration gradients.

The ibidi chemotaxis system provides answers to the following experimental questions:

• chemotactic activity
  
• strength of chemotactic activity
  
• influence of substances on chemotactical activity
  
• cell morphology and viability
  
• cell-cell interaction

µ-Slide Chemotaxis3D The µ-Slide Chemotaxis3D was developed for investigating the chemotactical behavior of non-adherent cells in gel matrices. It is possible to observe the migration in linear and stable concentration profiles for over 48 hours. As gradients can be rapidly established, a fast response (occurring in less than 30 minutes) can be measured.		µ-Slide Chemotaxis The µ-Slide Chemotaxis is optimized for analyzing the chemotactical response of adherent cells in linear and stable concentration profiles. Due to the gradient’s time stability of over 48 hours, it is the first system able to analyze chemotaxis of slow migrating cells for up to two days.

Gradient stability

The gradient formed in µ-Slide Chemotaxis provides a linear profile over the observation area. It is stable for at least 48 hours. Such a time period also allows slowly migrating cells to migrate significant distances.

Data analysis
“Chemotaxis and Migration Tool”

ibidi has developed a free software analysis tool to analyze data from chemotaxis experiments (time stacks).  This 'Chemotaxis and Migration Tool' now runs as an independent program which is available for free here. The tool provides different types of graphs and statistical tests to perform advanced analysis of experimental data.

After cell tracking, the cells’ paths can be plotted and analyzed for chemotactical effects. An easy microscopic calibration gives access to a variety of parameters. For specific analyses there are sector tools which are able to count cells in angular or circular areas (Rose Plots).  A statistical test for inhomogeneity of cell distribution (Rayleigh test) completes the software.

All calculations can be directly visualized by histograms and diagrams. Optionally, all data can be exported for further analysis. With a complete set of migrational data, the user is able to quantify chemotaxis and random migration.

Chemotaxis parameters

For quantification of chemotaxis and migration, several values can be generated by the software tool. The center of mass and the y and x Forward Migration Indices are a measure of directed cell migration. The directionality may not necessarily indicate a chemotaxis effect.

Center of mass (Mend) The center of mass represents the averaged point of all cell endpoints. Its x and y values indicate in which direction the group of cells primarily traveled.
x and y Forward Migration Indices (xFMI, yFMI) The xFMI and yFMI represent the efficiency of forward migration of cells relating to the x or y axis. The larger the index on an axis the stronger the chemotactic effect on this axis. For simplification it is assumed that either the x-axis or the y-axis are parallel to the direction of the chemotactic gradient.
Directionality (D)  The directionality is calculated by comparing euclidian and accumulated distance. It is a measure of directness of cell trajectories. A directionality of D -> 1 means a straight-line migration from start to endpoint.

Chemotaxis – example experiment

The following example experiment was performed with the chemotactic cell line HT1080 (human fibrosarcoma) with serum as a chemoattractant.

0 Experimental parameters

1) Preparation

2) Video microscopy

Video microscopy must be used when performing an ibidi chemotaxis and migration assay. Without video microscopy there is no access and analysis of chemotaxis effects.

3) Cell tracking

Cell tracking is the only way to access to quantification of cell movement between frames of a temporal stack. Typically, tracking is done manually or automatically by special tracking software. Automated tracking algorithms need distinct objects, i.e. fluorescent labeled cells. After tracking the cells' traces their (x, y) values are available for each point of time (t).

4) Plotting the data

After the coordinate transformation, all initial points are set to (0,0) automatically by the ibidi Chemotaxis and Migration Tool. Cell trajectory plots can easily be created. For better visualization of chemotaxis effects additional information can be added to the plots, e.g. color information of cells moving up/down.

5) Chemotaxis values

All desired chemotaxis values can easily be generated by the Chemotaxis and Migration Tool.

6) Interpretation of data

Simple interpretation

The simplest way to interpret data is a visual inspection of the cell trajectories (plots). Strong and significant chemotaxis effects towards one specific direction can be easily seen. Furthermore, significant differences in cell velocity and directionality between chemotaxis and control experiment can be distinguished. When dealing with strong chemotaxis effects, as seen in our example, or total chemotaxis inhibition; simple interpretation might be sufficient. We also recommend taking the provided Rayleigh test into account. In our example you can also see that the total amount of chemoattractant, without any gradient, does lead to a different migration behavior of the cells.

Advanced interpretation

In addition to the optical impression of the plots, profound parameters can be used to prove a chemotaxis effect or a hypothesis. Parameters like the displacement of the center of mass (M) or the forward migration indices are valid measures when they are compared to the right reference measurements. In our example experiment the y_FMI of the (+/-) measurement is significantly higher than the x_FMI and also than the x_FMI and y_FMI of reference measurements (-/-) and (+/+).

Downloads for manual tracking

For data analysis we developed a Chemotaxis and Migration Tool based on ImageJ.
The tool can be downloaded here.

For tracking cells from a time stack we recommend to use Manual Tracking: http://rsb.info.nih.gov/ij/plugins/track/track.html