3-3--dihexyl-2-2--oxacarbocyanine and 5-5--6-6--tetrachloro-1-1--3-3--tetraethylbenzimidazolocarbocyanine

Increased lymphocyte death is a hallmark of human immunodeficiency virus (HIV) infection. Although virological factors have been linked to this phenomenon, increased cell death rates are still observed in treated individuals in which viral replication is halted. To understand the nature of this remaining altered cell death, we have developed a simple and fast assay to assess major cell death pathways in lymphocytes isolated from HIV-infected individuals. The combination of three factors: (i) antibody staining to identify CD3(+) CD4(+) and CD3(+) CD8(+) cells, (ii) assessment of mitochondrial and plasma membrane function using DiOC6(3) or JC-1 probes and vital dyes, and (iii) caspase inhibition, allowed for the quantification of caspase-independent and -dependent cell death in CD4 and CD8 T cells. The latter mechanism was divided in intrinsic and extrinsic apoptotic pathways according to the sensitivity of the dissipation of mitochondrial membrane potential to Z-VAD-fmk or Q-VD-oPH treatment. Our data show similar results for both caspase inhibitors in treated infected individuals, whereas Q-VD-oPH showed a more potent inhibition in viremic individuals, yielding lower levels of intrinsic apoptosis. Comparison of DiOC6(3) and JC-1 probes yielded similar results in CD4 T cells, allowing for a clear definition of death mechanism in these cells. However, in CD8 T-cells, JC-1 showed heterogeneous staining and detected significantly lower levels of cell death with a higher contribution of intrinsic apoptosis. In conclusion, we provide a simple method to assess CD4 T-cell death mechanisms in HIV-infected individuals. The reasons and consequences of mitochondrial heterogeneity in CD8 T-cells require further evaluation.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Benzimidazoles; Carbocyanines; Case-Control Studies; Caspase Inhibitors; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Flow Cytometry; Fluorescent Dyes; HIV Infections; Humans; Necrosis; Propidium; Quinolines; Staining and Labeling

Sperm motility evaluation is associated with fertility in IVF programmes. The visual estimation of sperm motility is extremely subjective. Hence, alternative methods are required. Among them, determination of mitochondrial membrane potential (Deltapsi(m)) changes of spermatozoa using potentiometric dyes may be a reliable test to determine sperm quality. However, the use of the potentiometric dyes in sperm samples has not been compared.. We have studied sperm samples from 28 infertile patients enrolled in an IVF programme in flow cytometry after staining of spermatozoa with four commonly used potentiometric dyes. Sperm motility was evaluated visually.. As expected, JC-1 seems to detect specifically Deltapsi(m) changes, CMX-Ros, DiOC(6)(3) and TMRE fluorescence is easily analysed and the latter three fluorochromes are particularly suitable for multiparametric staining. Irrespective of the Deltapsi(m)-dependent fluorochromes used to stain spermatozoa, a positive correlation was found between the percentage of Deltapsi(m)(high) cells and forward motility and also with high fertilization rates after IVF.. The four fluorochromes may be useful for evaluation of sperm samples from infertile patients. The choice of the potentiometric dyes will depend on their fluorescence characteristics in order to use them in combination with other fluorescent markers.

Topics: Adult; Benzimidazoles; Carbocyanines; Cell Survival; Fertilization; Flow Cytometry; Fluorescent Dyes; Humans; Infertility, Male; Male; Membrane Potentials; Mitochondria; Organic Chemicals; Organometallic Compounds; Semen Preservation; Sensitivity and Specificity; Sperm Motility; Spermatozoa; Staining and Labeling

Release of cytochrome c, a decrease of membrane potential (Deltapsi(m)), and a reduction of cardiolipin (CL) of rat brain mitochondria occurred upon incubation in the absence of respiratory substrates. Since CL is critical for mitochondrial functioning, CL enrichment of mitochondria was achieved by fusion with CL liposomes. Fusion was triggered by potassium phosphate at concentrations producing mitochondrial permeability transition pore opening but not cytochrome c release, which was observed only at >10 mm. Cyclosporin A inhibited phosphate-induced CL fusion, whereas Pronase pretreatment of mitochondria abolished it, suggesting that mitochondrial permeability transition pore and protein(s) are involved in the fusion process. Phosphate-dependent fusion was enhanced in respiratory state 3 and influenced by phospholipid classes in the order CL > phosphatidylglycerol (PG) > phosphatidylserine. The probe 10-nonylacridine orange indicated that fused CL had migrated to the inner mitochondrial membrane. In state 3, CL enrichment of mitochondria resulted in a pH decrease in the intermembrane space. Cytofluorimetric analysis of mitochondria stained with 3,3'-diexyloxacarbocyanine iodide and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzymidazolylcarbocyanine iodide showed Deltapsi(m) increase upon fusion with CL or PG. In contrast, phosphatidylserine fusion required Deltapsi(m) consumption, suggesting that Deltapsi(m) is the driving force in mitochondrial phospholipid importation. Moreover, enrichment with CL and PG brought the low energy mitochondrial population to high Deltapsi(m) values and prevented phosphate-dependent cytochrome c release.

Topics: Acridine Orange; Animals; Benzimidazoles; Brain; Carbocyanines; Coloring Agents; Cyclosporine; Cytochrome c Group; Dose-Response Relationship, Drug; Flow Cytometry; Fluorescent Dyes; Hydrogen-Ion Concentration; Kinetics; Liposomes; Membrane Fusion; Membrane Potentials; Mitochondria; Phosphates; Phosphatidylglycerols; Phosphatidylserines; Phospholipids; Protein Binding; Rats; Time Factors

Maintenance of the mitochondrial membrane potential (Deltapsim) is fundamental for the normal performance and survival of cells such as cardiomyocytes, that have a high energy requirement. Measurement of Deltapsim is therefore essential in order to develop an understanding of the molecular mechanisms controlling cardiomyocyte function. Here we have evaluated various potentiometric dyes for their ability to detect alterations of Deltapsim, using flow cytometry and confocal microscopy.. Primary cultures of cardiomyocytes from neonate rats were treated with mitochondrial uncouplers before or after loading with Rho123, DiOC(6)(3), CMXRos or JC-1, and then analysed by flow cytometry. Apoptotic cells were identified by light scatter and Annexin V staining.. The four potentiometric dyes tested were able to discriminate between viable and apoptotic cells. However, only JC-1 was able to detect the collapse of Deltapsim induced by uncouplers of mitochondrial respiration. Confocal microscopic analysis confirmed that JC-1 stained mitochondria in a potential-dependent manner. In contrast, CMXRos stained cardiomyocytes irrespective of alterations in Deltapsim.. We conclude that JC-1 is the optimal dye to use when measuring Deltapsim in cardiomyocytes.

Topics: Animals; Apoptosis; Benzimidazoles; Carbocyanines; Cells, Cultured; Evaluation Studies as Topic; Flow Cytometry; Fluorescent Dyes; Immunohistochemistry; Intracellular Membranes; Membrane Potentials; Microscopy, Confocal; Mitochondria, Heart; Organic Chemicals; Rats; Rats, Sprague-Dawley; Rhodamine 123

The sensitivity and specificity of three fluorescent probes used for cytofluorimetric analysis of mitochondrial membrane potential (delta psi) were studied in the U937 human cell line. First, the role of plasmamembrane in influencing the binding of the probes to mitochondria has been investigated. The depolarization of plasmamembrane with high doses of extracellular KCl had no immediate effects on the loading of JC-1, DiOC6(3) and rhodamine 123 (R123). However, after a few hours of culture in the presence of KCl, significant changes were observed only in cells stained with DiOC6(3). Second, a comparative study was performed concerning the effects of agents capable of collapsing deltapsi. While adding FCCP to cell cultures resulted in consistent changes in the fluorescence emission of both JC-1 and DiOC6(3) - but not of R123 - only cells stained with JC-1 responded to valinomycin. On the whole, our data indicate that JC-1 is a reliable probe for analyzing delta psi changes with flow cytometry, while the others show a lower sensitivity (R123), or a non-coherent behaviour, due to a high sensitivity to changes in plasmamembrane potential [DiOC6(3)]. These data cast some doubts on those studies that, using fluorescent probes that have a low sensitivity to delta psi, hypothesized that the fall in delta psi is one of the early events, if not one of the main causes, of apoptosis.

Topics: Apoptosis; Benzimidazoles; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Fluorescent Dyes; Humans; Intracellular Membranes; Ionophores; Membrane Potentials; Mitochondria; Potassium Chloride; Reproducibility of Results; Rhodamine 123; Rhodamines; Tumor Cells, Cultured; Uncoupling Agents