Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry.

Leonarda Troiano, Roberta Ferraresi, Enrico Lugli, Elisa Nemes, Erika Roat, Milena Nasi, Marcello Pinti, Andrea Cossarizza

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)2719-2727
Number of pages9
JournalNature Protocols
Volume2
Issue number11
DOIs
Publication statusPublished - 2007

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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