carbocyanines and 1-1--(4-4-7-7-tetramethyl-4-7-diazaundecamethylene)bis-4-(3-methyl-2-3-dihydro(benzo-1-3-thiazole)-2-methylidene)quinolinium

Fluorescence Resonance Energy Transfer (FRET) is a powerful tool to determine distances between chromophores bound to macromolecules, since the efficiency of the energy transfer from an initially excited donor to an acceptor strongly depends on the distance between the two dye molecules. The structure of the noncovalent complex of double-strand DNA (dsDNA) with thiazol orange dimers (TOTO) allows FRET analysis of two intercalated chromophores. By intercalation of two different TOTO dyes we observe an energy transfer from TOTO-1 as donor and TOTO-3 as acceptor. In this manner we are able to determine the mean distance between two proximate TOTO molecules bound to dsDNA. Thus the maximum number of binding positions for this type of intercalation dyes in the dsDNA can be obtained. Furthermore the dependency of the acceptor emission on the donor concentration is analysed. The emission of TOTO-3 reaches a maximum when the acceptor-to-donor ratio is 1:10.

Topics: Carbocyanines; Dimerization; DNA; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Quinolines; Quinolinium Compounds; Thiazoles

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.. 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).. 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.. 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.

Topics: Apoptosis; Benzimidazoles; Carbocyanines; Cell Death; Cell Line; Cell Membrane; Fas Ligand Protein; Flow Cytometry; Fluorescent Dyes; Humans; Hydrogen Peroxide; Kinetics; Light; Membrane Glycoproteins; Membrane Potentials; Microscopy, Confocal; Mitochondria; Necrosis; Propidium; Quinolinium Compounds; Scattering, Radiation; Thiazoles; Time Factors

We examined five nucleic acid binding fluorescent dyes, propidium iodide, SYBR Green I, YO-PRO-1, TOTO-3, and TO-PRO-3, for nuclear DNA staining, visualized by fluorescence and laser confocal microscopy. The optimal concentration, co-staining of RNA, and bleaching speeds were examined. SYBR Green I and TO-PRO-3 almost preferentially stained the nuclear DNA, and the other dyes co-stained the cytoplasmic RNA. RNAse treatment completely prevented the cytoplasmic RNA staining. In conventional fluorescence microscopy, these dyes can be used in combination with fluorescence-labeled antibodies. Among the dyes tested, TOTO-3 and TO-PRO-3 stained the DNAs with far-red fluorescence under red excitation. Under Kr/Ar-laser illumination, TOTO-3 and TO-PRO-3 were best suited as the nuclear staining dyes in the specimens immunolabeled with fluorescein and rhodamine (or Texas red).

Topics: Animals; Benzothiazoles; Benzoxazoles; Carbocyanines; Cell Nucleus; Diamines; DNA; Fluorescent Antibody Technique; Fluorescent Dyes; Microscopy, Confocal; Microscopy, Fluorescence; Organic Chemicals; Propidium; Quinolines; Quinolinium Compounds; Rats; Rats, Sprague-Dawley; Ribonucleases; Thiazoles