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 tagging with directly or indirectly labeled nucleotides incorporated by the use of terminal deoxynucleotidyl transferase (TdT). Propidium iodide (PI) is typically added for the simultaneous assessment of DNA content. PI was found to quench the specific in situ FITC-fluorescence of apoptotic cells which were labeled by TdT with FITC-conjugated dUTP, biotin-dUTP followed by streptavidin-FITC, or digoxigenin-dUTP followed by FITC-labeled anti-digoxigenin antibodies as measured by flow cytometry. The effect was concentration-dependent, with 50% quenching occurring at 0.8 microg/ml, 1.5 microg/ml, and 5 microg/ml PI, respectively, at approximately 1 x 10(6) cells/ml. Spectrofluorimetry in solution revealed that 15 microg/ml PI was required to quench 50% of the fluorescence of ss FITC-labeled poly(dU)35. In contrast, the fluorescence of ds FITC-labeled poly(dU)35-poly(dA) was quenched to 50% at 3 microg/ml PI. The 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, and the fluorescence yield of PI at 610 nm increased, but the binding required 10-fold higher concentrations of poly(dU)35 as compared to ds DNA. The spectroscopic properties of PI are therefore similar whether bound 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 explained by direct binding of PI to the poly(dU) tails synthesized by TdT in situ in apoptotic cells, but may rather be due to radiationless energy transfer from FITC to PI bound to double-stranded DNA close to the nicks where TdT is known to start polymerization.