Combined Boyden

Leukocyte infiltration is a crucial event of inflammation. This process is regulated by several molecules, including chemoattractants and cellular adhesion molecules [1]. Chemoattraction is termed chemotaxis when the ligand gradient is soluble. Chemotaxis is described as an ensemble of cell activities that are precisely orchestrated in space and time, consisting of highly dynamic cell shape rearrangements accompanied by rapid adhesion–de-adhesion cycling and directional movement [2].

Different models for investigating leukocyte chemotaxis have been developed to measure chemotactic and random motility responses in vitro. These assays provide information on the locomotion of leukocytes, which is not only of physiological interest, but also important in furthering our understanding of the inflammatory response. Although these assays do not necessarily mimic in vivo conditions, they are useful tools for studying the cellular functions that occur in living tissues [3], [4], [5], [6].

The most common methods used to evaluate chemotaxis are those in which leukocytes migrate through a micropore membrane [7], of which the Boyden model is an early example [3]. Although this technique provides an estimation of the relative leukocyte chemotactic activity of soluble substances, it is suggested that it gives partial and sometimes misleading information, and should be supplemented with other methods including visual assays [8]. Consequently, the Boyden chamber technique has been modified over the years [9], [10], [11]; however, none of these modifications allow the quantification of total migrating leukocytes concurrently with the determination of their phenotype.

Phenotypification of migrating leukocytes is not required for chemotaxis studies that utilize purified leukocyte subsets, such as neutrophils [12] and monocytes [13]. However, in many experimental paradigms, the simultaneous evaluation of distinct leukocyte subsets is crucial [12], [14], [15], and this typically requires separate assays. To associate specific inflammatory responses with distinct leukocyte subsets, investigators require an improved method for studying the density and phenotype of diverse leukocyte subsets simultaneously. In the present study we describe and validate an improved CBFCA, which allows the accurate quantification and phenotypification of different migrating leukocyte subsets in a single assay. This CBFCA allows the accurate quantification and recovery of the migrating leukocytes for subsequent phenotypification and characterization in a single assay, which facilitates understanding of leukocyte trafficking during the inflammatory response.