Sensitive and reliable JC-1 and TOTO-3 double staining to assess mitochondrial transmembrane potential and plasma membrane integrity: Interest for cell death investigations

Abstract : Background Apoptosis is currently studied by flow cytometry with mitochondrial membrane potential (Δψmt) and membrane integrity fluorochromes. Rhodamine 123 and DiOC6(3) remain controversial to identify cells displaying a low Δψmt. JC-1 constitutes a good Δψmt indicator, due to a fluorescence shift from green to orange emission, according to the increase in Δψmt. Nevertheless, it is not feasible to analyze it simultaneously with propidium iodide. Among available fluorescent probes, TOTO-3 seems to be a good candidate for double staining with JC-1. Methods Cell death of HaCaT cells was induced by H2O2 and FasL. Samples were stained with DiOC6(3)/IP or JC-1/TOTO-3 then analyzed by flow cytometry. Results were supported by confocal microscopy analyses of mitochondrial membrane potential. Moreover, cell morphology was determined on the sorted subpopulations defined on the basis of staining (JC-1 versus TOTO-3). Results We found that JC-1 is a more efficient mitochondrial probe than DiOC6(3). After stress induction, the fluorescence level of JC-1 and TOTO-3 clearly defined three fluorescent subpopulations, respectively: (1) JC-1high and TOTO-3low, (2) JC-1low and TOTO-3medium, and (3) JC-1low and TOTO-3high. Their morphologic aspects after cell sorting indicated that they corresponded to three functional states (intact, apoptotic, and necrotic cells), and data were supported by caspase activity measurements. Conclusions We propose a reliable and efficient staining, with JC-1 and TOTO-3 to discriminate three functional cellular states: intact, apoptotic, and necrotic/late apoptotic cells by flow cytometry.
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https://hal.univ-lorraine.fr/hal-01656587
Contributeur : Raphaël Duval <>
Soumis le : mardi 5 décembre 2017 - 18:05:53
Dernière modification le : jeudi 1 mars 2018 - 11:53:40

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Thomas Zuliani, Raphaël E. Duval, Chantal Jayat, Sylviane Schnébert, Patrice André, et al.. Sensitive and reliable JC-1 and TOTO-3 double staining to assess mitochondrial transmembrane potential and plasma membrane integrity: Interest for cell death investigations. Cytometry Part A, Wiley, 2003, 54A (2), pp.100 - 108. 〈10.1002/cyto.a.10059〉. 〈hal-01656587〉

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