title: Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry.
abstract: The analysis of changes in mitochondrial membrane potential (MMP) that can occur during apoptosis provides precious information on the mechanisms and pathways of cell death. For many years, the metachromatic fluorochrome JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide) was used for this purpose. Thanks to new dyes and to the technical improvements recently adopted in several flow cytometers, it is now possible to investigate, along with MMP, a variety of other parameters. Using three sources of excitation and polychromatic flow cytometry, we have developed a protocol that can be applied to cells undergoing apoptosis. In the model of U937 cells incubated with the chemopreventive agent quercetin (3,3',4',5,7-pentahydroxyflavone), we describe the detection at the single cell level of changes in MMP (by JC-1), early apoptosis (exposition of phosphatidylserine on the plasma membrane detected by annexin-V), late apoptosis and secondary necrosis (decreased DNA content by Hoechst 33342 and permeability of the plasma membrane to propidium iodide). The procedure can be completed in less than 2 h.
authors.affiliation: Department of Biomedical Sciences, University of Modena and Reggio Emilia, via Campi 287, 41100 Modena, Italy.
authors.forename: Leonarda
authors.initials: L
authors.lastname: Troiano
authors.forename: Roberta
authors.initials: R
authors.lastname: Ferraresi
authors.forename: Enrico
authors.initials: E
authors.lastname: Lugli
authors.forename: Elisa
authors.initials: E
authors.lastname: Nemes
authors.forename: Erika
authors.initials: E
authors.lastname: Roat
authors.forename: Milena
authors.initials: M
authors.lastname: Nasi
authors.forename: Marcello
authors.initials: M
authors.lastname: Pinti
authors.forename: Andrea
authors.initials: A
authors.lastname: Cossarizza
mesh_terms: D017209:Apoptosis; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D011794:Quercetin / Q000494:pharmacology; D020298:U937 Cells
chemical_list: D011794:Quercetin