- 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?
- How many cells are being tracked during each acquisition?
- Could you explain the difference between Forward Migration Index (FMI) and the Displacement of Center of Mass?
- How do I characterize a chemotaxis effect?
- What is a gradient?
- How steep is the gradient? Which gradients can cells sense?
- Over time, how stable is the gradient?
- What chemoattractant should be used?
- Which concentration should I use as a chemoattractant?
- If all the holes on the µ-Slide Chemotaxis chamber are plugged, is it still permeable to the CO2?
- Does the steepness of the gradient change?
- How long do cells have to move directionally, before it is certain that it is not just a random walk?
- My cells do not migrate. What can I do?
- How can I do a Fibronectin coating in the µ-Slide Chemotaxis?
- 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?
- Is imaging of chemotaxis only possible with an inverted microscope?
- Do you have a video demo that shows how to use the µ-Slide Chemotaxis?
- I would like to do chemotaxis experiments with non-adherent cells. What is the necessary number of cells for this type of experiment?
- How many cell lines were reported to successfully migrate in a gel microenvironment?
- Is there any difference between the various types of collagens?
- Are there any charts available that show the dependence of the gradient steepness, from the concentration of the agent?
- Does the gel in the µ-Slide Chemotaxis affect diffusion and gradient establishment?
- I have a heterogeneous population of non-adherent cells that I want to test in a competitive migration assay, with two different chemokines. After the migration, I’d like to fix and permeabilize the cells and stain them. Can I use the µ-Slide Chemotaxis?
- I often have trouble loading the observation chamber with the gel mix. How can I improve my method?
- I have a problem with bubbles that appeared after loading cells in the middle slide of the µ-Slide Chemotaxis. Do you have any recommendations on how to solve this problem?
- Is there a simpler 2D high-throughput assay for the initial screening of chemoattractants?
- Is the ibiTreat surface suited for working with HUVECS, or should I coat it with something?
- I often have trouble loading cells into the observation chamber of the µ-Slide Chemotaxis. How can I improve my method?
- I have my chemotaxis data files saved either in a .tif format or an .avi format. Can you tell me which format is needed to start my analysis?
- What kind of data table is needed for chemotaxis data analysis?
- Is it possible to use data from other chemotaxis tracking programs?
- How can I plot my chemotaxis data table?
- What parameters should be used when presenting chemotaxis results?
- What algorithm is used by the Chemotaxis and Migration Tool to convert the tracks into Rose plot data?
- What factors should dictate how I set the interior angle?
- What is the range interval?
- I am interested in looking at the myelination of neurons in the peripheral nervous system (PNS) and the central nervous system (CNS). Will this chemotaxis software help as a tracking video, in 2D or 3D, in this process?
- Why do all cells start from just one point in your plots?
- What does the Rayleigh Test actually test?
- What is the difference between the Rayleigh Test and the Rayleigh Test for vector data?
- When calculating the p-value of the Rayleigh test, what image should be studied? Does the software calculate that for me?
- What is the difference between slice series and track series?
- Is it a problem when some data points are missing?
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.
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.
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).
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.
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.
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.
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.
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.
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.
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 (PDF).
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.
No, you can also use upright microscopes. With this setup, the working distance of the objective needs to be at least 14 mm.
Yes, please check out the following Movie: Chemotaxis Assays
I would like to do chemotaxis experiments with non-adherent cells. What is the necessary number of cells for this type of experiment?
For each chamber of the µ-Slide Chemotaxis, we use 6 µl of a cell suspension with a final concentration in the gel of 3x106 cells/ml. Therefore, 18,000 cells per chamber are necessary.
So far, we have tested the dendritic cells HT-1080 and HUVEC in the µ-Slide Chemotaxis. The results for all cell types were positive.
There are huge differences in protocol and handling, such as the variations between collagen derived from a rat’s tail and the kind derived from bovine collagen. Also, regardless of the origin, the gelation time (handling time) can also differ greatly.
Are there any charts available that show the dependence of the gradient steepness, from the concentration of the agent?
The concentration profile is always linear, from the used concentration to zero, whereas the shape of the concentration profile is independent of the used concentration.
Please find more detailed information on gradient measurements here.
Typical aqueous gels, such as collagen gels or Matrigel™, are thought to not hinder diffusion in the µ-Slide Chemotaxis. When using stiff hydrogels with pore sizes in the range of the diffusing molecules, this approach is invalid and no chemical gradient can be established inside the gel.
I have a heterogeneous population of non-adherent cells that I want to test in a competitive migration assay, with two different chemokines. After the migration, I’d like to fix and permeabilize the cells and stain them. Can I use the µ-Slide Chemotaxis?
To date, we have not tested the fixation of cells in gel inside the observation area of µ-Slide Chemotaxis. From tube formation assays, we generally know that fixation, permeabilization, blocking, and staining of cells on Matrigel™ is possible. Therefore, it should also be possible to do immunostainings in the 3D chemotaxis assay. In this case, we recommend removing the liquid from one reservoir and successively filling the second reservoir with the different solutions. We can assume that the incubation time should be increased by a factor of 4, in order to give the solutions sufficient time to diffuse into the observation area. The filling of liquids should be carefully done, in order not to push out the gel from the observation area.
I have a problem with bubbles that appeared after loading cells in the middle slide of the µ-Slide Chemotaxis. Do you have any recommendations on how to solve this problem?
The most common reason for air bubbles in the µ-Slide Chemotaxis is gas being released from the plastic, due to a temperature increase when placed into the incubator. We recommend degassing all liquids, and also the slide, before using them. Degassing is done be placing the slide and the medium inside of the cell culture incubator for 24 hours before you start the experiment.
No, there is no simpler assay. The only alternative, the Boyden assay, will not give you clean and actionable results. The 2D setup with the µ-Slide Chemotaxis from ibidi offers a better quality readout data and trustworthy readouts.
Normally, ibiTreat is fine with HUVEC, especially, when a growth medium containing 10% extra serum (fetal calf serum) is used. In µ-Slide Chemotaxis, we recommend using the Collagen IV pre-coated slides, because cell adherence is much faster and easier. Optionally, it is possible to do the coating on your own, by following Application Note 08.
I often have trouble loading cells into the observation chamber of the µ-Slide Chemotaxis 2D. How can I improve my method?
I have my chemotaxis data files saved either in a .tif format or an .avi format. Can you tell me which format is needed to start my analysis?
Before you can do a final analysis of your chemotaxis data with the Chemotaxis and Migration Tool, you need to track your images or movies. This can be either done by using Manual Tracking, an ImageJ plug-in, or with another software program.
ibidi’s Chemotaxis and Migration Tool can only analyze data, such as plotting cell trajectories and calculating values. It cannot track the cells. Our software requires a data table with tracks, slices, and x/y positions of the cells. Please find some example data, for initial use, on the Chemotaxis and Migration Tool website.
The data tables need to have a tab separated (t) format. For a detailed description, please refer to the instructions of the Chemotaxis and Migration Tool.
Yes, but the data needs to be in a special table format. The first row and column are necessary, and may contain arbitrary characters. All other rows must have the following tab separated (t) format:
For a detailed description, please refer to the instructions of the Chemotaxis and Migration Tool.
- Import an appropriate data table into the Chemotaxis and Migration Tool.
- Select the required number of slices (e.g., the number of pictures used for tracking). The number of slices can be found in your original data table (“Show original data”).
- Calibrate the software by setting the x/y pixel size and the time interval. The x/y calibration represents the edge length of a pixel in µm. The time interval represents the time between each slice.
- Press “Apply settings”, after changing the values and parameters.
- Plot trajectories (Plot data) and “export as image”.
- Export the values of FMI, center of mass, velocity, and Rayleigh test from the “Measured values” window.
Here are some recommendations for presenting the results (in talks or publications):
- Show the original movie (time lapse film of your cells).
- Show the trajectory movie (time lapse film + overlaid cell trajectories from the tracking).
- Display the trajectory plot (graph).
- Show the table or bar graph of center of mass, FMI, and Raleigh test.
What algorithm is used by the Chemotaxis and Migration Tool to convert the tracks into Rose plot data?
The histogram plot, rose diagram, circular plot, and density plot all use the same data. They are just plotted differently. Counting the cells in the different sectors generates the data.
See the following reference: Mardia Kanti V., Jupp Peter E., 1999, Directional Statistics, Wiley Series
What factors should dictate how I set the interior angle?
Use your personal taste, and also decide how detailed you want the data to be. The interior angle divides the whole circle into sectors.
The range interval determines how smooth the data points become. The smoothness is based on the size of the range interval, which in turn acts like a catchment area. Inside, the computer will be counting the end points.
I am interested in looking at the myelination of neurons in the peripheral nervous system (PNS) and the central nervous system (CNS). Will this chemotaxis software help as a tracking video, in 2D or 3D, in this process?
Currently, the Chemotaxis and Migration Tool is only designed for use with 2D setups. In the future, we also plan to analyze a setup in 3D. To help us with our development, please send us your image stacks and we will try to analyze them.
Plotting all cell trajectories using a coordinate transformation (setting all start points from x, y to 0, 0) is a common procedure in chemotaxis research. Of course, not all cells actually start from the very same point in the experiment, we just do this for better visualization.
The Rayleigh test is a statistical test of the uniformity of a circular distribution of points. The null hypothesis (uniformity) is rejected with p-values smaller than p=0.05.
The Rayleigh test is published here:
Fisher, N. I. (1993) Statistical Analysis of Circular Data, Cambridge University Press, New York.
The Rayleigh Test for vector data also takes into account the distance from origin, when creating an analysis.
When calculating the p-value of the Rayleigh test, what image should be studied? Does the software calculate that for me?
The Rayleigh test uses the last image (cell endpoint), and is automatically calculated by the software.
Slice series exports data from single slices (= time points). Track series exports data from different tracks (= 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 software rejects all tracks with missing data points. Even if only one data point is missing, there is a problem and the track is not used. Please check that each track is complete by opening your data table in Excel.