TIME-RESOLVED FLUOROMETRIC ASSAY FOR NATURAL-KILLER ACTIVITY USING TARGET-CELLS LABELED WITH A FLUORESCENCE ENHANCING LIGAND

A time-resolved fluorometric assay for the measurement of natural kill er cell activity against target cells labelled with the acetoxymethyl ester of the fluorescence enhancing ligand 2,2':6',2N-terpyridine-6,6' '-dicarboxylic acid (TDA) is described. The hydrophobic esterified for m of TDA (bis(acetoxymethyl)2,2':6',2 ''-terpyridine-6,6 ''-dicarboxyl ate, BATDA) diffuses readily through the cell membrane of viable cells . BATDA is hydrolysed by intracellular esterases resulting in accumula tion of membrane impermeable TDA inside the target cells. After incuba tion of labelled K-562 cells with effector cells the TDA released from lysed cells into the supernatant is chelated with Eu3+. The natural k iller cell activity is then quantified by measuring the intense fluore scence of the EuTDA chelates formed. Target cells are rapidly labelled when incubated with BATDA, TDA is released from target cells faster t han Cr-51, the spontaneous release permits a short-term release assay to be set up and the detection of EuTDA is fast (5 min/96 well plate). Furthermore, this non-radioactive method permits the use of complex c ulture media since, in contrast to methods based on prompt fluorometry , the problem with autofluorescence can be avoided by the use of time- resolved fluorometry.