MOLAR QUANTIFICATION BY FLOW-CYTOMETRY OF FATTY-ACID-BINDING TO CELLSUSING DIPYRROMETHENEBORON DIFLUORIDE DERIVATIVES

Fatty acid analogs of a dipyrrometheneboron difluoride fluorophore (BD Y-FA) have recently been developed. Relative to other fluorescent fatt y acids, some of these have the advantages of excitation and emission spectra similar to those of fluorescein and of high quantum yield, whi ch permits their use in conventional argon laser cytometry or microsco py. For the cytofluorimetric quantification of BDY-FA analogs, express ed as molecules bound per cell, we have compared the fluorescence of B DY-dodecanoic acid (BDY-C-12) with that of fluorescein. Fluorescent be ads with different amounts of bound fluorescein were used to calibrate a flow cytometer in order to correlate the fluorescence intensity wit h the number of fluorescein molecules per bead, In addition, starting from the basic equation defining the relationship between fluorescence and concentration, we have derived another equation which makes it po ssible to establish, for a given fluorescence, the relative molar conc entration of both fluorochromes and, consequently, to express the fluo rescence intensity emitted by the BDY-FA as the equivalent number of B DY-FA molecules. As an example of the potential application of this pr ocedure, the time-course and concentration-dependent binding of BDY-C- 12 to quiescent and mitogen-activated human lymphocytes and to culture d human T-lymphoma cells have been studied. The method described is of general interest as it can also be applied to the now cytometric or l aser scanning microscopic quantification of other fluorescent dyes. (C ) 1996 Wiley-Liss, Inc.