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Sensitive and reliable JC‐1 and TOTO‐3 double staining to assess mitochondrial transmembrane potential and plasma membrane integrity: Interest for cell death investigations
Author(s) -
Zuliani Thomas,
Duval Raphaël,
Jayat Chantal,
Schnébert Sylviane,
André Patrice,
Dumas Marc,
Ratinaud MarieHélène
Publication year - 2003
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.10059
Subject(s) - propidium iodide , biology , flow cytometry , rhodamine 123 , microbiology and biotechnology , apoptosis , programmed cell death , cell sorting , biochemistry , multiple drug resistance , antibiotics
Background Apoptosis is currently studied by flow cytometry with mitochondrial membrane potential (Δψ mt ) and membrane integrity fluorochromes. Rhodamine 123 and DiOC 6 (3) remain controversial to identify cells displaying a low Δψ mt . JC‐1 constitutes a good Δψ mt indicator, due to a fluorescence shift from green to orange emission, according to the increase in Δψ mt . 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 H 2 O 2 and FasL. Samples were stained with DiOC 6 (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 DiOC 6 (3). After stress induction, the fluorescence level of JC‐1 and TOTO‐3 clearly defined three fluorescent subpopulations, respectively: (1) JC‐1 high and TOTO‐3 low , (2) JC‐1 low and TOTO‐3 medium , and (3) JC‐1 low and TOTO‐3 high . 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. Cytometry Part A 54A:100–108, 2003. © 2003 Wiley‐Liss, Inc.