For establishing a positive control for a chemotaxis effect, we recommend using chemoattractants that have already been published with your cell type of interest, if available. If nothing is known, we advise using fetal calf serum for initializing a chemotaxis effect.

The concentration of a chemoattractant depends greatly on the used cell type and the experimental setting. For initial experiments, we recommend doing a literature research for the chemoattractant of interest, for example publications using Transwell assays. If no concentrations have been published, we advise screening a range of concentrations in 10-fold steps, for example 1 nM, 10 nM, 100 nM, and 1 µM, to identify the most effective chemotaxis result.

For each chamber of the µ-Slide Chemotaxis, we recommend seeding 6 µl cell suspension in a gel matrix with a concentration of 3 × 10⁶ cells/ml. Thus, the calculated number of cells per chamber is 18,000.

In order to get statistically significant data without overcrowding the observation area, ca. 200 cells should be present in the observation area of the µ-Slide Chemotaxis when starting the imaging. For more details on the cell concentration, please read Application Note 17.

Yes. The mimicking of in vivo-like migration of non-adherent cells through interstitial tissue is a good argument for using gels in migration assays. We developed a protocol for investigating chemotactic migration of non-adherent dendritic cells in a Collagen I gel. Please refer to Application Note 23 for further details.

HUVECs generally adhere well when using ibidi labware with the ibiTreat tissue culture-treated surface, especially when a growth medium containing 10% fetal calf serum is used. To speed up cell adhesion during chemotaxis assays, however, we recommend using µ-Slides Chemotaxis precoated with Collagen IV. Optionally, it is possible to coat the slides yourself by following the protocol in Application Note 08.

Coating the µ-Slide Chemotaxis with fibronectin is easy. A general coating protocol can be found in Application Note 08.

Using an inverted microscope is the most convenient method for imaging a chemotaxis experiment using the µ-Slide Chemotaxis. However, you can also use upright microscopes, but with this setup, the working distance of the objective needs to be at least 14 mm.

The ibidi chemotaxis assay is no endpoint assay. It is impossible to see or count the differences between start and end in a visual assay with homogeneous start conditions, because most cells migrate with low directness. Therefore, video microscopy and cell tracking are necessary.

Please find more information in the application chapter “Experimental Workflow of a Chemotaxis Assay”.

We recommend observing the cell migration for at least 10 times the cell diameter. This is only a rough estimation, but you get representative trajectories. Shorter trajectories might not be representative, while much longer trajectories do not add more migratory information.

Example: HT-1080 cells are approximately 20–50 µm in diameter and their average speed is ca. 40 µm/hour. This means that after 10 hours, you get representative trajectories for most single cells in that population. In other words, most cells moved about 10 times their diameter.

Please check out the following video for an overview on how to pipet the µ-Slide Chemotaxis: Chemotaxis Assays Using the µ-Slide Chemotaxis. Find more detailed information about the planning, procedure, and data analysis of chemotaxis assays here.

The gradient establishes within the first few hours after the chemoattractant has been added into the µ-Slide Chemotaxis. After that, an equilibrium is reached and the gradient does not change for the next 48 hours. After that, the gradient slowly flattens due to molecule transport across the observation area.

Please find an example of a gradient measurement on the µ-Slide Chemotaxis product page.

The ibidi µ-Slide Chemotaxis is designed to provide a quick gradient with excellent long-term stability for more than 48 hours. In 3D chemotaxis assays using aqueous gels, such as Collagen I or Matrigel®, the gradient is stably established and not influenced by the gel in any way.

Typical aqueous gels, such as collagen gels or Matrigel®, do not interfere with diffusion of a chemoattractant molecule in the µ-Slide Chemotaxis. The pore sizes of the gels are too large to affect diffusing molecules. Whenever a gel matrix pore size is smaller than the diffusing molecules, diffusion will be impaired and a gradient will not be established in a regular way.

When performing chemotaxis assays, the terms gradient and concentration profile are often confused with each other.

A concentration profile describes the distribution of a concentration, for example of a specific compound or chemoattractant, over a certain distance. The gradient is the slope at one particular point of this concentration profile.

In a linear concentration profile, the gradient, or slope, is identical at all positions, whereas the absolute concentration changes along the distance. When there are no concentration differences, the gradient is 0.

In chemotaxis assays, cells respond to concentration differences of a chemoattractant rather than to the absolute concentration alone. Therefore, understanding the difference between the concentration profile and the gradient is important for interpreting directed cell migration.

For controlled and reproducible gradient generation, specialized systems such as the µ-Slide Chemotaxis can be used to establish stable and defined concentration profiles.

Usually, it is sufficient to track 30–40 cells per observation field. However, when there is a weak chemotaxis effect, it might be necessary to track more cells to obtain statistical significance. For a strong chemotaxis effect, it might be sufficient to track fewer than 20 cells per condition.

Here are some recommendations for presenting the results in talks or publications:

• Show the original video, meaning the time-lapse film of your cells.
• Show the trajectory video, meaning the time-lapse film with overlaid cell trajectories from the tracking.
• Display the trajectory plot.
• Show the table or bar graph of the Center of Mass, FMI, and Rayleigh test.

Please find detailed information about the data analysis and interpretation of chemotaxis assays in the application chapter “Data Analysis of Chemotaxis Assays”.

The Rayleigh test is a statistical test for the uniformity of a circular distribution of points, meaning cell endpoints.

With p < 0.05, the null hypothesis, uniformity, is rejected, indicating a chemotaxis effect. Like all statistical tests, this one strongly depends on the number of analyzed cells.

Moore BR., 1980, A modification of the Rayleigh test for vector data, Biometrika, Volume 67, 175–180.

The Rayleigh test for vector data also includes the distance from the origin.

When using the Chemotaxis and Migration Tool, all coordinates of the cell trajectories are transformed for a better visualization when creating the plot: all start points are set from x, y to 0, 0. This is a common procedure in chemotaxis analysis. Of course, not all cells actually start from the very same point in the experiment.

When using the Chemotaxis and Migration Tool, slice series exports data from single slices, meaning time points. Track series exports data from different tracks, meaning cells. For example, Slice Series / Directness exports the parameter Directness of each time point, averaged. Track Series / Directness exports the parameter Directness of each cell.

The Chemotaxis and Migration Tool automatically uses the last image, meaning the cell endpoint, for calculating the p-value of the Rayleigh test.

For a detailed description on how to plot chemotaxis data tables when using the Chemotaxis and Migration Tool, please refer to the Chemotaxis and Migration Tool Instructions.

The Chemotaxis and Migration Tool software rejects all tracks with missing data points. Even if only one data point is missing, the track is not used. Therefore, please check that each track is complete, for example with Excel.

Before you can do a final analysis of your chemotaxis data with the Chemotaxis and Migration Tool, you need to track the cells using the acquired images or videos. The Chemotaxis and Migration Tool requires a tab-separated data table with tracks, slices, and x/y positions of the cells. This can be created by using Manual Tracking, an ImageJ plug-in.

Datasets from Manual Tracking can be imported directly into the Chemotaxis and Migration Tool software. For a detailed description, please refer to the Chemotaxis and Migration Tool Instructions.

To practice every step of the analysis, find Example Data of a Chemotaxis Assay.

Cell migration is a very sensitive process, which is influenced by many factors such as the substrate, coating, as well as the medium and its supplements. If the cells migrate very slowly or do not migrate at all, we suggest trying different experimental conditions, changing only one condition at a time.

The migrating ability also depends strongly on the cell type, cell density, and passage, as all this can influence whether the cells are in a migratory or non-migratory state. In addition, some cell types need special stimuli or have to undergo differentiation before they are able to migrate. Before using a new cell type for a chemotaxis assay, a literature research on these factors is recommended.

Yes, the polymer material of the µ-Slide Chemotaxis is still gas permeable, even after closing all filling ports with the plugs. This gas permeability, however, is low when the chamber is completely closed. This means that it will take quite a long time for the CO₂ and O₂ to seep into the liquid when the slide is inside the incubator. To avoid air bubbles, we recommend degassing all liquids and the slide before using them by placing them inside the incubator for 24 hours before starting the experiment.

The most common reason for air bubbles in the µ-Slide Chemotaxis is gas being released from the medium and the polymer, which is due to a temperature increase when the slide is placed in the incubator. To avoid this, we recommend degassing all liquids and the slide before using them by placing them inside the incubator for 24 hours before starting the experiment.

Please refer to the detailed troubleshooting section in Application Note 17.

Yes. The fixation, permeabilization, blocking, and staining of cells inside gel matrices is easily possible. For the protocol, please refer to Application Note 44.