Chemotaxis Boyden chamber

Cells respond to some extracellular factors such as leukotriene B (Ford-Hutchin-son et al., 1980) by increasing the locomotion rate in an undirected manner as opposed to chemotaxis. This mechanism, known as chemokinesis, is likely on purely statistical grounds to result in cells accumulating at the site of origin of this stimulus (Wilkinson, 1987). The differentiation of factors that are chemotactic from chemokinetic responses can be difficult, but this has been greatly facilitated using the Boyden chamber (Lackie, 1986). 

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 experiments (see Note 2) Use blind well Boyden chambers, that contain a volume of 100 pL in the lower compartment (Costar, Bodenheim, Germany). As filters use selfpunched (7 pm punch) polyvinylpyrrolidone containing polycarbonate filters (pore size 3 mm, Costar), which prior to use need to be washed with 1 M NaOH in 50 % (v/v) aqueous ethanol for 7 min followed by three washes in water (see Note 3). Use p-nitrophenyl-p-D-glucuronide (Sigma) at 10 mM in 0.1 M aqueous sodium acetate, pH 4.0, for p-glucuronidase detection (see Note 4). 

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 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. 

To determine the optimal concentration for collecting the invasive population, it is often necessary to perform dose response experiments and try a range of chemoattractant amounts. We have found that for EGF, the optimal concentration in the in vivo invasion assay is 25 nM while for the in vitro Boyden chamber chemotaxis it is 5 nM. The difference can be explained by different diffusion properties of the gradient emanating from a tip of a needle into live tissue vs. EGF in a buffer being directly accessible to migrating cells across pores in a filter. By the same token, the optimal... 

The possibility that G glycoprotein mediated FKN-like chemotactic responses was examined using modified Boyden chambers. Leukocytes from naive BALB/c mice, or leukocytes from normal human adults, were tested for chemotaxis toward FKN or G glycoprotein. Both FKN and G glycoprotein induced similar leukocyte chemotaxis that was inhibited with antibodies specific... 

Chemokines promote the directional migration of cells expressing CKRs, which can be measured in chemotaxis assays using a Boyden chamber. Cells placed in the upper chamber, which is separated with a porous... 

Pelz, G., Schettler, A., and Tschesche, H., Granulocyte chemotaxis measured in a Boyden chamber assay by quantification of neutrophil elastase. Eur. J. Clin. Chem. Clin. Biochem. J. 1993, 31 651 56. 

Put 4 x 10s cells are 28 uL of medium containing 1% BSA in each well in the lower compartment of the 48-well Boyden Chemotaxis chamber. 

Nind, A.E, Neutrophil chemotaxis technical problems with nitroceUulose filters in Boyden-type chambers. /. Immunol. Meth., 1981,49 39-52. 

---