Cytokines and chemokines chemotaxis

Most cytokines have been identified thanks to appropriate bioassays, which have also provided tools to measure these mediators and to standardize them (3). Even when immunometric methods are available, bioassays are invaluable tools to assess the functional relevance of immunoreactive material and to quantitate undefined mediators.

Here we will focus on chemotaxis, the eponimous function of the chemokine superfamily (4, 5). N-terminal processing of chemokines results in products with reduced activity or with a different spectrum of action, thus emphasizing the importance of assaying function (4-6).

Chemotaxis is defined as the directional locomotion of cells sensing a gradient of the stimulus. Chemotaxis has been extensively studied with leukocytes that are 'professional migrants', but a variety of cell types including fibroblasts, melanoma cells, keratinocytes, and vascular endothelial cells exhibit directional locomotion in vitro. Two main techniques have been used to measure migration in vitro: migration under agarose and chemotaxis across porous membranes. While the former approach may more closely resemble the in vivo conditions, the latter is easier to quantitate and allows analysis of directional versus random locomotion. We will therefore focus on the description of migration through a porous membrane. Both a classic modified Boyden chamber assay (7) and a micromethod (8, 9) will be described. Protocol 1 describes the use of a micro-method for assessing leukocyte chemotaxis. A schematic representation of the micro chemotactic chamber is shown in Figure 1.

Isolation Monocytes Protocol

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LOWER COMPARTMENT OF THE CHAMBER

POROUS FILTER SILICON TRIMMING

UPPER COMPARTMENT OF THE CHAMBER

Figure 1 Schematic representation of the 48-well micro Boyden Chemotaxis chamber.

Protocol 1

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