A Rapid, Quantitative Fluorescence Assay for Cell Damage by Cytotoxic Antibodies

Abstract Our laboratory recently (1) described a fluorochromatic assay for cell damage by cytotoxic alloantibody, which was based on retention of fluorescein by intact cells after its hydrolysis from fluorescein diacetate esters (FDA) (2). Although the method gave reproducible results in our hands, it was slow and always subject to error, since traces of complement used in the reaction were capable of hydrolyzing FDA, thus yielding erroneously high values of cell viability. This was not satisfactory and accordingly we sought another approach to differential fluorescent staining of cells, which would still allow machine-reading of the assays. Rather than looking for other compounds that were enzymatically changed from non-fluorescent to fluorescent, we chose to screen a number of fluorescent probes (3), compounds whose fluorescence is enhanced when bound to protein or nucleic acids. Such compounds were initially screened by observing fresh and heat-damaged lymphocytes, bathed in solutions of various probes, with the fluorescence microscope.