Functional changes during apoptosis revealed by the simultaneous detection of mitochondrial membrane potential and DNA content by JC-1 and Hoechst 33342

ApopThe study of functional changes that occur in intracellular organelles such asmitochondria during cell death/apoptosis are of consistent interest since severalyears. Probes exist that allow the analysis of a variety of mitochondrial (mt)parameters, including mt mass and mt membrane potential (MMP), along withthose that stain DNA and reveal its modifications. The dye JC-1, excited by theargon laser present in almost all flow cytometers, is considered the gold standardfor detecting MMP (Current Protocols in Cytometry, 9.14.1-9.14.7, 2000), andemits in FL-1 (monomers) and FL-2 (aggregates, whose formation is due by ahigh MMP). However, its spectrum of emission does not allow the use of otherdyes excitable by such laser since there is a relevant signal also in FL-3. Thus,until now, the simultaneous study of changes in DNA content and MMP duringapoptosis have been difficult if not impossible, and all data, including ours(Cytometry, 40: 189-197, 2000) have been obtained by indirect measures. In orderto investigate such parameters in the same cell, we took advantage of a novelinstrument, i.e. CyFlow ML by Partec (Germany), equipped with an air-cooledargon ion laser (488 nm, 200 mW), a UV Mercury lamp HBO (100 long life, 100W), a red diode laser (635 nm, 25 mW), a Nd:YAG laser (532 nm, 50 mW) and aCCD camera. Such configuration allowed us to use JC-1, excited by the Ar laser,along with Hoechst 33342 excited by the UV lamp. Apoptosis was induced inU937 or in CEM cell lines by a 48 hour incubation with 50 μM quercetin. Theanalysis of MMP allowed us to identify 3 different populations, which correspondedto three different stages of cell death. The first (about 50% of cells) hada normal MMP (high red and intermediate green fluorescence by JC-1), normalDNA content, FSC and SSC. In the second population (about 20%), MMP waschanging (high red and high green fluorescence), DNA content was normal whileFSC and SSC were those of apoptotic cells. In the third population (about 30%),MMP was consistently decreased (low red and high green fluorescence), DNAcontent was decreased (with the presence of the typical hypodiploic peak), FSCand SSC were altered. Thus, our data indicate that, in this model of apoptosis,cells start to loose MMP (as JC-1 aggregates begin to become monomers, causingan increase in green fluorescence) and change their physical parameter beforethe beginning of loss of nuclear DNA and the appearance of the hypodiploic peak.Studies are in course for the simultaneous detection of changes in mt mass (measuredby Mitotracker Deep Red) or other parameters (such as plasma membranepermeability or phosphatidylserine exposure) with dyes excited by the red laser.