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

A microplate-based fluorometric assay was developed for quantitation of can cer 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 l ine MDA-MB-231. For the assay, bovine cortical bone from the shaft of the f emur was cut, turned, and sliced to B-mm-diameter round 200-mu m-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 flu orometer 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) c ontaining the H+/K+ ionophore nigericin, which equilibrates internal and ex ternal pH. By taking advantage of the pH sensitivity of BCECF, the fluoresc ence intensity in the assay was increased 2.5 times compared to cells loade d with calcein. The specificity of the assay was demonstrated with a specif ic immuneserum raised against the MDA-MB-231 cell line. The assay can be co mpleted in 1 h and permits the use of a large number of samples and is ther efore useful for screening potential drug candidates that could block cance r cell attachment to bone material. Moreover, the assay described can easil y be used to characterize molecular structures involved in cell attachment to bone. (C) 1999 Academic Press.