Diffusion gradient assay

Figure 1 Schematic of a chemotaxis experiment. "A" shows a diffusion gradient assay, and "B" shows a transendothelial chemotaxis assay.

When the pH is different on the two sides of the membrane, the transport of ioniz-able molecules can be dramatically altered. In effect, sink conditions can be created by pH gradients. Assay improvements can be achieved using such gradients between the donor and acceptor compartments of the permeation cell. A three-compartment diffusion differential equation can be derived that takes into account gradient pH conditions and membrane retention of the drug molecule (which clearly still exists—albeit lessened—in spite of the sink condition created). As before, one begins with two flux equations... 

Under agarose. This assay was originally developed to study leukocyte migration (Nelson et al., 1975) however, it has also been applied to the study of chemotactic and chemokinetic effects of FGF on endothelial cells (Stokes et al., 1990). Cells are allowed to migrate under an agarose gel in which a chemoattractant (or a control solution) forms a diffusion gradient. The differential migration of cells toward the chemoattractant is taken as a measure of its chemotactic activity. 

As seen in Fig. 1, two main modalities can be distinguished. Cell migration in diffusion gradient chemotaxis assays is mainly dependent on the strength of the chemokine-receptor interaction, but transendothelial... 

Conventional free interface diffusion achieves high transient levels of supersaturation, but has a complicated spatial/temporal gradient due to the constant cross-section of the capillary. This gradient couples the kinetics and thermodynamics of traditional free interface diffusion assays in a way that pFID does not. 

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... 

Commercial tube diffusion or multi-plate assays are by nature simple to use and do not require the use of expensive instrumental equipment. Over the years some improvements have been realized. For example, the incubation step is typically performed using a water bath. However, the use of water baths has some disadvantages. Temperature gradients can occur within the unit (unless the water is adequately... 

Assays in which a gradient of the stimulant is estabiished by diffusion... 

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