Chemotaxis assay

Chemotaxis assays are experimental tools for evaluation of chemotactic ability of prokaryotic or eukaryotic cells. A wide variety of techniques have been developed. Some techniques are qualitative - allowing an investigator to approximately determine a cell's chemotacic affinity for an analyte - while others are quantitative, allowing a precise measurement of this affinity.

In general, the most important requisite is to calibrate the incubation time of the assay both to the model cell and the ligand to be evaluated. Too short incubation time results no cells in the sample, while too long time perturbs the concentration gradients and measures more chemokinetic than chemotactic responses.

The most commonly used techniques are grouped into two main groups:

This way of evaluation deals with agar-agar or gelatine containing semi-solid layers made prior to the experiment. Small wells are cut into the layer and filled with cells and the test substance. Cells can migrate towards the chemical gradient in the semi solid layer or under the layer as well. Some variations of the technique deal also with wells and parallel channels connected by a cut at the start of the experiment (PP-technique). Radial arrangement of PP-technique (3 or more channels) provides the possibility to compare chemotactic activity of different cell populations or study preference between ligands.

Counting of cells: positive responder cells could be counted from the front of migrating cells, after staining or in native conditions in light microscope.

Chambers isolated by filters are proper tools for accurate determination of chemotactic behavior. The pioneer type of these chambers was constructed by Boyden. The motile cells are placed into the upper chamber, while fluid containing the test substance is filled into the lower one. The size of the motile cells to be investigated determines the pore size of the filter; it is essential to choose a diameter which allows active transmigration. For modelling in vivo conditions, several protocols prefer coverage of filter with molecules of extracellular matrix (collagen, elastin etc.) Efficiency of the measurements was increased by development of multiwell chambers (e.g. NeuroProbe), where 24, 96, 384 samples are evaluated in parallel. Advantage of this variant is that several parallels are assayed in identical conditions.

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