PROPIDIUM IODIDE QUENCHES THE FLUORESCENCE OF TDT-INCORPORATED FITC-LABELED DUTP IN APOPTOTIC CELLS

Apoptotic cells with frequent DNA strand breaks may be detected, by ta gging with directly or indirectly labeled nucleotides incorporated by the use of terminal deoxynucleotidyl transferase (TdT), Propidium iodi de (PI) is typically added for the simultaneous assessment of DNA cont ent. PI was found to quench the specific in situ FITC-fluorescence of apoptotic cells which were labeled by TdT with FITC-conjugated dUTP bi otin-dUTP followed by streptavidin-FITC, or digoxigenin-dUTP followed by FITC-labeled anti-digoxigenin antibodies as measured by flow cytome try, The effect was concentration-dependent, with 50% quenching occurr ing at 0.8 mu g/ml, 1.5 mu g/ml, and 5 mu g/ml PI, respectively, at ap proximately 1 x 10(6) cells/ml. Spectrofluorimetry in solution reveale d that 15 mu g/ml PI was required to quench 50% of the fluorescence of ss FITC-labeled poly(dU)(35). In contrast, the fluorescence of ds FIT C-labeled poly(dU)(35)-poly(dA) was quenched to 50% at 3 mu g/ml PI. T he maximum of the fluorescence excitation spectrum of PI shifted from 490 nm to 535 nm upon binding to ds DNA as well as ss poly(dU)(35), an d the fluorescence yield of PI at 610 nm increased, but the binding re quired 10-fold higher concentrations of poly(dU)35 as compared to ds D NA, The spectroscopic properties of PI are therefore similar whether b ound to poly(dU) or to double-stranded DNA, but the binding to poly(dU ) is much weaker. The observed quenching in situ therefore cannot be e xplained by direct binding of PI to the poly(dU) tails synthesized by TdT in situ in apoptotic cells, but may rather be clue to radiationles s energy transfer from FITC to PI bound to double-stranded DNA close t o the nicks where TdT is known to start polymerization. (C) 1998 Wiley -Liss,Inc.