### What is the recommended number of cells that need to be plated in a µ-Slide Chemotaxis, in order to get statistically significant data, but not overcrowd the observation area?

The goal is having ca. 200 cells in the observation area of the µ-Slide Chemotaxis. The recommended initial concentration is 3 x 106 cells/ml. We suggest analyzing 40 cells per image for statistical significance. In the case that there is a weak chemotactic response, even more cells should be tracked.

### How many cells are being tracked during each acquisition?

We track 30 to 40 cells. When there is a weak chemotaxis effect, it might be necessary to track more cells to obtain a statistical significance.

### Could you explain the difference between Forward Migration Index (FMI) and the Displacement of Center of Mass?

The FMI is very similar to the Displacement of Center of Mass. However, to calculate the FMI, the x- and y- components of each single cell are divided by the total length the cell traveled through the experiment.

### How do I characterize a chemotaxis effect?

The chemotactic potential is derived from the values of the Forward Migration Index (FMI) and the Rayleigh test. For data interpretation, the FMIs of both the chemotaxis experiments and the control experiments are compared to one another. In the case of chemotaxis, all FMI components of the control experiments (-/-, +/+) and the perpendicular FMI of the chemotaxis experiment (+/‑) should be around 0. Values that are significantly different than 0 will represent a chemotaxis effect. The Rayleigh test p values are compared to each other to test for homogeneous distributions.

The terms gradient and concentration profile are often confused with each other. The concentration profile describes the distribution of concentration over a certain distance. In linear concentration profiles, the gradient (slope) is identical at all positions, whereas the absolute concentration changes. The gradient is the slope on one point of a concentration profile. When there are no concentration differences, the gradient is 0.

For the µ-Slide Chemotaxis, the concentration drops over a distance of 1 mm. The injected concentration always drops to 0 across the channel.

Cells can sense both gradient and absolute concentrations. They sense grad c / c (grad c = local gradient of concentration, and c =concentration at this point).

### Over time, how stable is the gradient?

After achieving equilibrium, the stability will last for approximately 48 hours, depending on the diffusion in the µ-Slide Chemotaxis. The rate of diffusion depends on three things: molecule size, dynamic viscosity, and temperature.

### What chemoattractant should be used?

We recommend using attractants for your cell type, which are already known and used in published literature. In the case that there is nothing known, we advise fetal calf serum for initializing a chemotaxis effect.

### Which concentration should I use as a chemoattractant?

For initial experiments we recommend using concentrations known from published literature (e.g., from the Transwell assays). In the case that there is nothing known, we advise screening concentrations over an entire order of magnitude using a factor of 1.000 (e.g., from 1 nM to 1 µM) to find out where an effective chemotaxis effect can be observed.

### If all the holes on the µ-Slide Chemotaxis chamber are plugged, is it still permeable to the CO2?

Yes, the plastic is still gas permeable, even after complete sealing the µ-Slide Chemotaxis chamber with the plugs. This gas permeability, however, is slow 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 inside the incubator. To prevent this, pre-incubate the liquid and the chamber before filling the chamber. By doing this, you can maintain optimal conditions for the cells the whole time. Also, always use a gas- and temperature-equilibrated medium and plastic material.

### Does the steepness of the gradient change?

Only during gradient establishment, which occurs 1 hour after the chemoattractant is filled into a µ-Slide Chemotaxis. After that time, equilibrium is reached.

### How long do cells have to move directionally, before it is certain that it is not just a random walk?

We recommend observing the cell migration for, at least, 10 times the cell diameter. This is only a rough estimation, but you get good, representative trajectories.

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

### My cells do not migrate. What can I do?

Sometimes a coating, or certain medium conditions, can cause this. It is well known that some cell types and coatings can have a huge impact on the ability and speed of cell migration. So, we suggest trying a different substrate, or conducting the experiment under different medium conditions.

How can I do a Fibronectin coating in the µ-Slide Chemotaxis?

Coating the µ-Slide Chemotaxis is not complicated. We do not recommend doing it for the first trials, unless that specific surface is crucial. Please keep in mind that after coating, the chamber needs to be completely dry to ensure that the filling procedure works. The general coating protocol can be found here.

In the corresponding Application Note 17, coating information is also included.

### Why are video microscopy and cell tracking needed for the chemotaxis assay? Can’t I simply count migrated cells at the end of the experiment?

The cell distribution, in the observation area, is homogeneous. After a chemotaxis effect, this distribution of cells changes slightly, and usually towards a higher chemoattractant concentration. It is impossible to see, or count, the differences between the start and end in a visual assay with homogeneous start conditions. Therefore, video microscopy is an absolute necessity. Additionally, tracking the cells is necessary, only because most cells migrate with low directness and it is hard to see a chemotaxis effect from the time-lapse movie.

### Is imaging of chemotaxis only possible with an inverted microscope?

No, you can also use upright microscopes. With this setup, the working distance of the objective needs to be at least 14 mm.