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Keywords:

  • polychromatic flow cytometry;
  • mitochondrial membrane potential;
  • apoptosis;
  • JC-1;
  • propidium iodide;
  • Hoechst;
  • Annexin-V

Abstract

Background

Until now, the simultaneous analysis of several parameters during apoptosis, including DNA content and mitochondrial membrane potential (ΔΨ), has not been possible because of the spectral characteristics of the commonly used dyes. Using polychromatic flow cytometry based upon multiple laser and UV lamp excitation, we have characterized cells with different ΔΨ during apoptosis.

Methods

U937 cells were treated with the flavonoid quercetin (Qu) and stained with JC-1 to detect ΔΨ, propidium iodide (PI) for cell viability, Hoechst 33342 for DNA content, Annexin V conjugated with Alexa Fluor-647 for detection of phosphatidilserine (PS) exposure, marker of early apoptosis, or Mitotracker Deep Red for the determination of mitochondrial mass.

Results

Treatment with Qu provoked the onset of three cell populations with different ΔΨ: (1) healthy cells, with normal ΔΨ, DNA content and physical parameters, high mitochondrial mass, PI- and Annexin V-negative; (2) cells with intermediate ΔΨ and normal DNA content, but with physical parameters typical of apoptotic cells and low mitochondrial mass; most of them were PI+ and Annexin V+; (3) cells with collapsed ΔΨ that had low mitochondrial mass and were Annexin-V+, PI+; half of them showed diminished DNA content. Similar results, i.e. the presence of cells with intermediate ΔΨ, were observed in other models of apoptosis.

Conclusions

During Qu-induced apoptosis, loss of ΔΨ, PS exposure, and decrease of mitochondrial mass are early events that precede permeability to PI and loss of DNA. Populations of cells with different ΔΨ, as revealed by flow cytometry after JC-1 staining, differed also for other parameters associated to apoptosis. Thus, the simultaneous analysis of several parameters by polychromatic flow cytometry permits a better identification of many stages of cell death, and, more in general, allows to evaluate the eventual heterogenic sensibility of the population under study to a given compound. © 2005 Wiley-Liss, Inc.