Chemotaxis Assay

Migration of cells in response to stimuli is responsible for several physiological functions mostly in the case of inflammatory responses and immune functions. The transwell chemotaxis assay is useful to study mechanisms of migration during chemotaxis. The main purpose of this assay is to determine if a molecule of interest exhibits chemotactic activity. Molecules that 

Load lOOtxL (1 x 10 cells) of cell suspension from each sample to the upper well or Inserts of the 24-well transwell plates. 

Place the Inserts or the upper well on the bottom well carefully, avoiding formation of air bubbles. 

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A series of triazolophthalazines were recently reported to be PDE2 inhibitors with IC50 values as low as 0.1 nM. In this series, compound 2 demonstrated activity in chemotaxis assays. No data were reported for effects on cyclic nucleotide levels, however, efficacy in cellular assays such as the chemotaxis assay suggests the effect is related to PDE2 inhibition in the cell types studied [12]. Pyridopyrimidines such as 3 were claimed to have IC50 values less than 50 nM [13]. 

Fig. F.2 Cartoon depiction of the transweii piate used for chemotaxis assay.

The reagents listed in Subheading 2. referencing this note are not needed if a chemotaxis assay is not performed. 

Purification of chemokines from natural sources requires special strategies. First of all, the detection system should be useful to detect the required chemokine at low concentrations, i.e., it is important to choose an assay system that allows the detection of low amounts of the chemokine in a screening system. Screening systems often used are Boyden chamber chemotaxis assay... 

Chemotaxis assays provide a very sensitive indicator of cellular mobilization and can easily be mastered by anyone with average laboratory experience. Several critical steps must be followed in loading a chemotaxis chamber. The first is to add the correct volume of chemokine to the bottom wells of the chamber. Typically, 26 pL is the correct volume to be loaded. Too small a volume results in air bubbles in the wells, while too large a volume overflows the wells. 

We generally assess the protein concentration of new chemokine stock solutions by OD280 using the appropriate extinction coefficient for each chemokine or by Bradford assay. Although this is not often practical when very small quantities of chemokine are purchased because 10-20% of the material may be sacrificed for an accurate determination, we have found that the actual protein concentration may vary by as much as 2CM-0% between lots which can significantly effect the reproducibility of chemotaxis assays. 

Use of an endothelial cell line should not be considered a substitute for primary endothelial cells such as HUVEC, as different endothelial cell lines may vary in the type and level of adhesion molecules expressed as well as their response to various stimuli such as cytokines. Endothelial cell lines provide a convenient, homogenous, and defined (if not necessarily relevant) cell mono-layer through which leukocytes must migrate in the chemotaxis assay. We find that use of an endothelial cell line to reduce the background migration of primary cells not only improves the signal-to-noise ratio of the assay but also the reproducibility of the assay as compared to HUVEC. 

Multi-well modified Boyden chambers can be obtained from Neuro Probe Inc. (Gaithersburg, MD). Model AP48 has been widely used for endothelial cell chemotaxis assays. The apparatus consists of top and bottom acrylic plates, a silicon gasket and assembly screws. The bottom plate has 48 wells, each with 25 pL final volume. These correspond to holes on the top plate, and form the upper wells when the chamber is assembled. The filter (polycarbonate, 25 x 80 mm) is placed between the top and bottom plates, and a gasket is placed over the filter to create the seal. The apparatus can be purchased with a selection of accessories, such as curved forceps, filter clamps, and wipers. These are required to process the filters after use. Filters can also be obtained from Nucleopore Inc. (Pleasanton, CA) and Costar (Cambridge, MA). 

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. 

Two types of filters are available polycarbonate and cellulose nitrate. Polycarbonate filters are used for endothelial chemotaxis assays. These filters are sided, with a matt and a shiny surface. The cells are allowed to adhere to the shiny surface prior to migration, and then stimulated to migrate to the matt surface. 

However, there are a number of points to consider when carrying out endothelial cell chemotaxis assays. 

Falk, W Goodwin, R. H and Leonard, E. J. (1980) A 48-well micro chemotaxis assay for rapid and accurate measurement of leukocyte migration. J. Immunol. Methods 33, 239-247. 

Protein extraction To isolate biologically active protein, organs can be placed in 1 mL cold 50 mM Tris-HCl, pH 7.5, 0.2 mM EDTA and homogenized on ice. Extracts are clarified by centrifugation at 15,000 for 30 min, followed by a second centrifugation at 100,000 for 45 min. This results in a preparation of protein which, in the case of chemokines, can be used for in vitro chemotaxis assays to determine bioactivity. The presence of transgenic protein in these preparations can also be documented by immunoblot. 

CCR3 Receptor Structure. Chimera studies of CCR3 and CCRl receptors show that the N-terminal segments of CCR3 appear to be important for eotaxin binding (77). However, eotaxin remained an effective agonist at this chimeric receptor in either calcium flux or chemotaxis assays. These data are consistent with a multisite model for chemokine-chemo-kine receptor interaction. 

Cells respond to chemical gradients. Depending on the concentration of the chemical, motile cells will move at different speeds towards or away from the gradient, depending on whether the chemical is an attractant or repellent. Microfluidic devices can go beyond traditional chemotaxis assays and enable the quantification of the cell s transport parameters. 

Table 1 summarizes the advantages and disadvantages of established chemotaxis assays, including microfluidic methods. In the following sections, we discuss methods for developing two new types of microfluidic chemotaxis systems (1) a pPlug model... 

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