Centrifugal assay for fluorescence-based cell adhesion adapted to the analysis of ex vivo cells and capable of determining relative binding strengths

Cell adhesion assays are widely used to identify novel cellular ligands, novel cell surface receptors for these ligands and to elucidate the mechanisms responsible for the underlying cellular and molecular interactions. We report here the development of a novel centrifugal assay for fluorescence-based cell adhesion (CAFCA) that offers a number of advantages over the currently available assays. CAFCA is based on two centrifugation steps: one to allow for the synchronization of the initial cell-substratum contact and one to enable both a defined removal force to be exerted onto the cells for displacement of unbound cells and determination of the relative binding strengths of adhering cells. The fluorescently tagged cells are monitored in specifically devised, disposable microplate assemblies by a two-sided fluorescence detection through the computer-interfaced SPECTRAFLUOR microplate fluorometer. The assay is rapid, accurate, reproducible and adaptable to small numbers of delicate primary cells that can ideally be labeled with the fluorochrome calcein AM (or analogous vital fluorescent dyes). Most uniquely, CAFCA provides (i) means of assessing the precise number of cells bound to a given substratum out of the total amount of cells contained within the population to be analyzed and (ii) a means of establishing the attachment strengths (i.e., dynes/cell) in a high number of samples/conditions simultaneously. CAFCA is therefore expected to make a substantial methodological and conceptual contribution to the range of available assays aimed at examining cellular interactions in vitro and promises the potential of being able to transpose automated versions of these tests for routine use in laboratories.