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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.

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Figure 1: Optical bench and filters utilized in the study.
Figure 2: JC-1 staining of cells with a low MMP.
Figure 3: Gating strategy for the analysis of cells with a different MMP during apoptosis by using a polychromatic flow cytometry protocol.
Figure 4: Physical parameters and MMP in Hoechstlow/PI− events.
Figure 5: Hoechst 33342 is better excited by the violet laser than by the UV lamp.

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Acknowledgements

This study has been partially supported by grants from Istituto Superiore di Sanità (Rome, Italy) to A. Cossarizza (Progetto Nazionale AIDS, Grant 30G.62). Partec GmbH (Münster, Germany) and Space Import-Export srl (Milan, Italy) are kindly acknowledged for continuous technical support and precious technical suggestions. Dr. Gabriele Marcotullio is acknowledged for excellent editorial assistance.

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Correspondence to Andrea Cossarizza.

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Troiano, L., Ferraresi, R., Lugli, E. et al. Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry. Nat Protoc 2, 2719–2727 (2007). https://doi.org/10.1038/nprot.2007.405

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