Chemotaxis chamber method

In a generalized Adler method, one end of a capillary tube (1 Xl disposable micropipette, 3 cm long with an internal diameter of 0.2 mm) is flame sealed. The entire capillary tube is then quickly passed through a flame, and while warm, the open end is plunged into a reservoir containing the test chemical dissolved in chemotaxis medium. The liquid is drawn up into the capillary as it cools and the filled capillary is then withdrawn from the reservoir and inserted into a chemotaxis chamber, which is constructed by placing a U-shaped melting point capillary tube between a microscope slide and a coverslip (Fig. 1.2). The chamber is filled with an appropriate chemotaxis medium and inoculated with bacteria so that the final concentration is approximately 6 x 10 cells/ml. After a 1-h incubation, the capillary is removed from the chamber and the exterior rinsed with sterile water. The sealed end of the capillary is then broken and the contents are... 

The advantages of the capillary assay are its simplicity, quantitative nature, and high sensitivity. Alternative methods for studying chemotaxis such as the swarm plate method of Adler (1966) require that the chemoattractant be metabolized. This is not necessary in the standard capillary assay. In addition, due to the small size of the chemotaxis chamber, only small amounts of compound are required to perform the experiments. The main disadvantage of this method is that the compound tested must be soluble in the chemotaxis medium. 

Willey and Waterbury (1989) used a substantially different method to study chemotaxis in the marine cyanobacterium Synechococcus sp. This experiment involved the use of blind-well chemotaxis chambers (Neuroprobe, Inc., Cabin John, MD) that consisted of an upper (800- il) and lower (200- li1) acrylic chamber separated by a polycarbonate filter (3.0 pm). A cell suspension (165 pi) of the cyanobacterium was placed in the lower chamber over which the polycarbonate filter was placed. An air space was left between the cell suspension and the filter to control the starting time of the experiment. The upper chamber was filled with sterile seawater containing the compound to be tested and then inverted, allowing the cell suspension to contact the polycarbonate filter and the seawater/compound solution. The experiments were run for 65 min, after which time the chambers were inverted to stop the experiment. The number of cells crossing the filter into the seawater chamber was determined by direct cell counts using epifluorescence microscopy. The motile strain of Synechococcus sp. tested in this assay elicited positive chemotaxis to compounds such as ammonia, nitrate, urea, glycine, and P-alanine. Control chambers with the same concentration of chemoattractant in both the upper and lower chambers failed to elicit a chemotactic response. While the compounds tested in this study were relatively simple metabolites, one could... 

The method used to perform the chemotaxis assay on endothelial cells is essentially that previously described for neutrophils and leukocytes, with one major difference. With neutrophils the cells are placed in suspension in the upper well and the chemotactic agent is placed in the lower well. However, with endothelial cells, the cells are placed in suspension in the lower well, and the filter and upper well put in place. The apparatus is then inverted, and left in an incubator for 2-4 h to allow the cells to adhere to the filter. After this period of incubation, the chambers are inverted again, back to their original orientation. The medium in the upper well is then replaced with fresh medium containing the chemotaxin, and experiment is allowed to proceed. 

The Boyden chamber is a simple apparatus used to test for chemotaxis, especially of leukocytes. It can also be used to assess tumor cell transmigration across an endo-thehal monolayer in vitro. It consists of two compartments separated by a MiUi-pore filter (3-8 pm pore size). A chemotac-tic factor is placed in one compartment, and a gradient develops across the thickness of the filter (ca. 150 pm). Cell movement into the filter is measured after an incubation period less than the time taken for the gradient to decay. Cell motility can be measured in Boyden chambers containing filters precoated with different materials, for example fibronectin or fibronectin fragments. The method, when apphed to malignant and non-mahgnant cell hnes, shows that the variable invasive potentials of these cells correlate with their abihty to disrupt the endothelial cell monolayer. 

Haddox, J. L., Pfister, R. R., and Sommers, C. 1. (1991) A visual assay for quantitating neutrophil chemotaxis in a collagen gel matrix. A novel chemotactic chamber. /. Immunol. Methods 141,41-52. 

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