Simultaneous Analysis of Relative DNA and Glutathione Content in Viable Cells by Phase-resolved Flow Cytometry

Background: Analysis of the DNA cell cycle and glutathione content cannot be performed on viable cells, because the fluorescence emissions of the DNA-specific probe Hoechst 33342 and the glutathione-specific probe monobromobimane overlap completely. We decided to explore whether the emissions could be resolved by the singlet excited state lifetimes of the probes.

Methods: Viable cells were first incubated with Hoechst 33342 at 37 degrees C for 30 min and then with monobromobimane at room temperature for 10 min. Samples were excited with a sinusoidally modulated laser beam (10 MHz) in a flow cytometer. The Hoechst 33342 and monobromobimane lifetimes and fluorescence intensities were resolved by using phase-sensitive detectors.

Results: The observed singlet excited state lifetimes were 1.5 ns for Hoechst 33342 and 12 ns for monobromobimane. The glutathione (GSH) content was shown to increase as cells (GM130, HL60, U937) progressed through the cell cycle. However, after the data were corrected for differences in cell volume, it was found that the GSH concentration was constant throughout the cell cycle of the exponentially growing cells.

Conclusions: Phase-resolved flow cytometry provides a means for the specific analysis of the GSH content/concentration as a function of the cell's position in the DNA cell cycle in viable cells.