Sensitive and reliable JC-1 and TOTO-3 double staining to assess mitochondrial transmembrane potential and plasma membrane integrity: interest for cell death investigations.
BACKGROUND
Apoptosis is currently studied by flow cytometry with mitochondrial membrane potential (Deltapsimt) and membrane integrity fluorochromes. Rhodamine 123 and DiOC6(3) remain controversial to identify cells displaying a low Deltapsimt. JC-1 constitutes a good Deltapsimt indicator, due to a fluorescence shift from green to orange emission, according to the increase in Deltapsimt. 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.
Zuliani
Thomas
T
LVMH-Recherches, Laboratoires R & D, Branche Parfums-Cosmétiques, Saint-Jean de Braye Cedex, France. thomas.zuliani@unilim.fr
Duval
Raphaël
R
Jayat
Chantal
C
Schnébert
Sylviane
S
André
Patrice
P
Dumas
Marc
M
Ratinaud
Marie-Hélène
MH
D017209:Apoptosis; D001562:Benzimidazoles / Q000494:pharmacology; D002232:Carbocyanines / Q000494:pharmacology; D016923:Cell Death; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D053222:Fas Ligand Protein; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008027:Light; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D009336:Necrosis; D011419:Propidium / Q000494:pharmacology; D011806:Quinolinium Compounds / Q000494:pharmacology; D012542:Scattering, Radiation; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors
D001562:Benzimidazoles; D002232:Carbocyanines; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D011806:Quinolinium Compounds; D013844:Thiazoles; C075295:1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-thiazole)-2-methylidene)quinolinium; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D011419:Propidium; D006861:Hydrogen Peroxide