A microplate-based fluorometric assay for monitoring human cancer cell attachment to cortical bone

A microplate-based fluorometric assay was developed for quantitation of cancer cell attachment to bone matrix. To evaluate this model system we used a panel of human cancer cell lines, including the human breast cancer cell line MDA-MB-231. For the assay, bovine cortical bone from the shaft of the femur was cut, turned, and sliced to 6-mm-diameter round 200-microm-thin disks and placed into the wells of a 96-well microplate. A fluorescent dye 2', 7'-bis(2-carboxyethyl-5(6)-carboxyfluorescein (BCECF-AM) and a microplate fluorometer equipped with a standard filter set for fluorescein isothiocyanate were used to detect the cells attached to the bone disks. The fluorescence was enhanced during the measurement step by using a K+ solution (pH 8.0) containing the H+/K+ ionophore nigericin, which equilibrates internal and external pH. By taking advantage of the pH sensitivity of BCECF, the fluorescence intensity in the assay was increased 2.5 times compared to cells loaded with calcein. The specificity of the assay was demonstrated with a specific immuneserum raised against the MDA-MB-231 cell line. The assay can be completed in 1 h and permits the use of a large number of samples and is therefore useful for screening potential drug candidates that could block cancer cell attachment to bone material. Moreover, the assay described can easily be used to characterize molecular structures involved in cell attachment to bone.