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Types of Chemotaxis Assays
2D Versus 3D Chemotaxis
It is commonly accepted that cells in a culture behave very differently when they are attached to a flat 2D surface, compared to being inside a 3D gel matrix. In most cases, the 3D environment mimics the in vivo situation much better. Gradients for chemotaxis can easily be built up in water-based gels (like Collagen I gels and Matrigel), because the gel structure does not hinder diffusion. The µ-Slide Chemotaxis is ideally suited for experiments in 2D and 3D environments.
Example of HT-1080 cancer cells on a 2D surface (top), and embedded into a Collagen I gel (bottom)
Typically, three to five repeat experiments are sufficient to create significant data from chemotaxis and -/- and +/+ control experiments. To achieve this, each experiment should contain data from 20 - 40 single cells, which is possible using low-magnification microscopy objective lenses, such as 5x or 10x.
Typical results of a 2D chemotaxis experiment with HT-1080 cells migrating towards fetal calf serum. The FMI (forward migration index) values parallel (║) and perpendicular (┴) to the gradient are shown. Significant chemotaxis is only observable parallel to the gradient in the +/- experiment. All other values are not significantly different from 0.
Chemotactic behavior of cells and mutants (knockout / knockdown)
Chemotactic potential of substances
Effect of chemotaxis inhibitors / enhancers
Distinguishing between chemotaxis and chemokinesis (enhanced migration)
Molecular mechanisms during chemotaxis, visualized by fluorescence microscopy
Investigation of signal transduction or cytoskeletal effects during chemotaxis
All examples are carried out in the µ-Slide Chemotaxis, either with cells attached to the 2D surface or embedded in a 3D gel matrix, respectively.
HUVEC Chemotaxis Inhibition
Endothelial cells, which tend to migrate in a collective manner, can be tested on compounds that inhibit chemotaxis functionality, even when there is a gradient present (e.g., compounds suppressing cytoskeleton dynamics, or receptor antagonists).
Fast leukocytes, such as dendritic cells or T-cells, can be visualized well during chemotaxis, when inside gel matrices like collagen. This is especially true when using high-resolution fluorescence microscopy, which shows changes in the cytoskeleton during immune response and 3D interstitial migration.
Chemotaxis, described as the directed motion towards a chemoattractant, can be distinguished from chemokinesis, which is known as an enhanced migration effect. Unlike transwell assays, the µ-Slide Chemotaxis allows to analyze both effects independently of each other. In the example, the chosen chemoattractant induces both chemotaxis and chemokinesis.
The ibidi μ-Slide Chemotaxis can additionally be used for various cell-to-cell chemotaxis assays. For example, cells brought inside the large reservoirs can be used as a chemoattractant producer.