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, no vel cell surface receptors for these ligands and to elucidate the mechanism s responsible for the underlying cellular and molecular interactions. Ne re port here the development of a novel centrifugal assay for fluorescence-bas ed cell adhesion (CAFCA) that offers a number of-advantages over the curren tly available assays. CAFCA is barred on two centrifugation steps: one to a llow 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 di splacement of unbound cells and determination of the relative binding stren gths of adhering cells. The fluorescently ragged cells are monitored in spe cifically devised, disposable microplate assemblies by a two-sided fluoresc ence defection through the computer-interfaced SPECTRAFLVUOR microplate flu orometer: The assay is rapid, accurate, reproducible and adaptable to small numbers of delicate primary cells that can ideally be labeled with the flu orochrome 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 Be pop ulation to be analyzed and (ii) a means of establishing the attachment stre ngths (i.e., dynes/cell) in a high number of samples/conditions simultaneou sly. CAFCA is therefore expected to make a substantial methodological and c onceptual 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 .