Assays of neutrophil motility

In animals, accumulation of neutrophils in the peritoneal cavity after injection of a stimulus is often used as an in vivo indicator of migration (e.g., [135, 213, 337, 341, 429]). In vivo assays for human studies utilize a skin chamber that is plaeed over a portion of disrupted skin accumulation of cells in the ehamber is then quantified [15, 325]. Whereas these assays are dependent upon the migratory capabilities of the cells, they also depend upon the ability of cells to adhere to the endothelium and mechanisms for clearance of cells from these compartments. Thus these assays are not uniquely migration assays and do not give detailed information about the characteristics of cell movement. 

Nonmotile neutrophils have a spherical morphology. Within a few minutes of activation with a chemoattractant (either in a gradient or in a uniform concentration), they develop the characteristic polar morphology of a lamellipodium at one end and a uropod at the other. This polarization is a prerequisite for migration of leukocytes, and thus measures of cell polarization are often used as indicators of migration [125, 170]. 

Mass of actin in detergent-soluble and -insoluble cytoskeleton 

Polarization is a prerequisite for neutrophil migration thus this assay is a [125, 170] measure of migratory potential, but not a direct measure of motility. 

Neutrophil motility requires significant rearrangement of the actin [289, 391] 

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An additional indirect assay of neutrophil motility is the actin polymerization assay. Chemoattractants induce a rapid, transient actin polymerization response in neutrophils (reviewed in [289, 391]), and since actin polymerization is required for migration to occur, it is often used as an indication of chemotactic capability. Several approaches have been developed to measure this response (reviewed in [289, 391]) (Table 2). Most commonly, cells in suspension are mixed with a putative chemoattractant then fixed and stained with a fluorescent phaUotoxin that binds to polymerized actin, but not monomeric actin. The bound fluorescence is quantified using flow cytometry or spectrofluorometry. The actin polymerization response to many neutrophil chemoattractants is rapid, reaching a maximum within 10 s of stimulus addition at 37° C. As with the cell polarization assay, no information about the migratory properties of the cells is obtained with the actin polymerization assay, only the likelihood of chemotactic activity is assessed. Like the cell polarization assay, the actin polymerization assay is useful for initial screening of chemoattractants, but must be followed up with an actual measurement of motility. 

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