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). 

More functional studies that address the process of migration use the so-called Boyden chamber [48]. In this assay the migratory capacity of endothelial cells after activation with chemoattractants or pro-angiogenic stimuli is studied. Velocity of migration and the percentage of cells that are capable of migrating through an ECM-coated membrane into another compartment can be determined. 

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). 

Fill the lower part of the Boyden chamber with appropriately treated HPLC fractions (see Note 22). 

Screw the upper part of the Boyden chamber tight to the lower part. 

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 method adapted for endothelial cells has proven to be simple, relatively robust, and reproducible in a number of laboratories. There are several variations on the basic method which have been reported in the literature. However, the precise method ultimately adopted will depend on... 

Two compartments Boyden chambers (commercially available as transwell filter plates). Fixative and cell staining solutions as described below microscope for visual and semiquantitative analysis titertek multiwell reader, or spectrophotometer for quantitative analysis. 

The Boyden-chamber chemoinvasion sissay, developed by Albini et al. (1987) using Matrigel , is an alternative simplified model system for measuring cell capacity to attach to extracellular matrix, degrade it, and migrate towards a chemoattractant. Matrigel is a... 

Dilute Matrigel in cold distilled water to 50 /xg in 50-100 /xl. Apply 25-50 /xg Matrigel to the filters, dry under hood, and reconstitute with serum-free medium. Place a solution of chemoattractant in the lower compartment of the Boyden chamber (in the absence of chemoattractant, very little cell migration occurs over a 6-h period). Place the coated filters in Boyden chambers, and close the chamber. Add to the upper chamber 2 to 3 x 10 cells in appropriate medium containing 0.1% BSA. Incubate at 37°C in 5% CO2 for 4-6 h. Remove the cells from the upper surface of the filter by wiping with a cotton swab or by passing them on tissue paper. Fix the filter (methanol or ethanol) and stain (haematoxylin-eosin or toluidine blue). Count the cells from various areas of the lower filter surface. Alternatively, migrated cells can be solubilized... 

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. 

B42. Bignold, L. P., Ferrante, A., and Haynes, D. R. Studies of chemotactic, chemotactic movement-inhibiting and random movement-inhibiting effects of interleukin-1 alpha and beta, tumour necrosis factors alpha and beta and interferon gamma on human neutrophils in assays using sparse-pore polycarbonate (Nuclepore) membranes in the Boyden chamber. Int. Arch. Allergy Appl. Immunol. 91, 1-7 (1990). 

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... 

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