carbocyanines and 3-3--dipentyl-2-2--oxacarbocyanine

Elimination of leukemia stem cells (LSCs) is necessary for the destruction of malignant cell populations. Owing to the very small number of LSCs in leukemia cells, xenotransplantation studies are difficult in terms of functionally and pathophysiologically replicating clinical conditions of cell culture experiments. There is currently a limited number of lead compounds that target LSCs. Using the LSC-xenograft zebrafish screening method we previously developed, we found that the fluorescent compound 3,3'-dipentyloxacarbocyanine iodide (DiOC5(3)) selectively marked LSCs and suppressed their proliferation in vivo and in vitro. DiOC5(3) had no obvious toxicity to human umbilical cord blood CD34+ progenitor cells and normal zebrafish. It accumulated in mitochondria through organic anion transporter polypeptides that are overexpressed in the plasma membrane of LSCs, and induced apoptosis via ROS overproduction. DiOC5(3) also inhibited the nuclear translocation of NF-κB through the downregulation of LSC-selective pathways, as indicated from DNA microarray analysis. In summary, DiOC5(3) is a new type of anti-LSC compound available for diagnostic imaging and therapeutics that has the advantage of being a single fluorescent chemical.

Topics: Animals; Apoptosis; Carbocyanines; Cell Line; Cell Proliferation; Fluorescent Dyes; Humans; Leukemia, Myeloid, Acute; Mitochondria; Neoplastic Stem Cells; Reactive Oxygen Species; Zebrafish

Neural progenitor cells and developing neurons show periodic, synchronous Ca(2+) rises even before synapse formation, and the origin of the synchronous activity remains unknown. Here, fluorescence measurement revealed that the membrane potential of the nuclear envelope, which forms an intracellular Ca(2+) store, changed with a release of Ca(2+) and generated spontaneous, periodic bursts of fluctuations in potential. Furthermore, changes in the nuclear envelope's potential underlay spike burst generations. These results support the model that voltage fluctuations of the nuclear envelope synchronize Ca(2+) release between cells and also function as a current noise generator to cause synchronous burst discharges.

Topics: Action Potentials; Animals; Calcium; Carbocyanines; Chick Embryo; Fluorescence; Fluorescent Dyes; Neural Stem Cells; Neurogenesis; Nuclear Envelope; Periodicity

Perfluorinated (aliphatic) acids (PFAs) and congeners have many applications in various industrial fields and household for decades. Years later they have been detected in wildlife and this has spurred interest in environmental occurrence as well as influencing living organisms. PFAs were established as peroxisome proliferators and hepatocarcinogens. Amphipatic structure suggests that they may alter cell membrane potential (mbDeltaPsi) and/or induce changes in cytosolic pH (pHi). The aim of this study was to examine the correlation between changes of above parameters and PFAs structure (CF(6)-CF(12)) in human colon carcinoma HCT116 cells. mbDeltaPsi and pHi were measured by flow cytometry using fluorescence polarization of the plasma membrane probe 3,3'-dipentyloxacarbocyanine (DiOC(5)(3)) and fluorescein diacetate (FDA), respectively. Dose- and time-dependent manner analysis revealed relatively fast depolarization of plasma membrane and acidification of cytosol both positively correlated with fluorocarbon chain length. mbDeltaPsi depletion after 4 h of incubation reached 8.01% and 30.08% for 50 muM PFOA and 50 muM PFDoDA, respectively. Prolonged treatment (72 h) led to dramatic dissipation of membrane potential up to 21.65% and 51.29% and strong acidification to pHi level at 6.92 and 6.03 at the presence of above compounds, respectively. The data demonstrate that PFAs can alter plasma membrane protonotrophy with the mode dependent on the compound hydrophobicity.

Topics: Carbocyanines; Carcinogens, Environmental; Cell Membrane; Cytosol; Dose-Response Relationship, Drug; Fatty Acids; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Fluorocarbons; HCT116 Cells; Humans; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Membrane Potentials; Molecular Structure; Structure-Activity Relationship; Time Factors

Dielectrophoresis (DEP) was used to examine a panel of MCF-7 cell lines comprising parental MCF-7 cells and MDR derivatives: MCF-7TaxR (paclitaxel-resistant, P-glycoprotein (P-gp) positive), MCF-7DoxR (doxorubicin-resistant MRP2 positive) plus MCF-7MDR1 (MDR1 transfected, P-gp positive). MCF-7DoxR and MCF-7MDR1 were broadly cross-resistant to natural product anticancer agents, whereas MCF-7TaxR cells were not, contrary to P-gp expression. Whilst DEP revealed modest membrane changes in MDR sub-lines, we saw significant changes in their cytoplasmic conductivity: MCF-7TaxR

Topics: Anthracyclines; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cell Survival; Cisplatin; Colchicine; Cytoplasm; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Electrophoresis; Etoposide; Female; Fluorescent Dyes; Humans; Inhibitory Concentration 50; Membrane Potentials; Membrane Transport Proteins; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Paclitaxel; Phenotype

In a previous study, we reported a new method for counting quail blood cells. After quail blood cells were stained with fluorescent lipophilic dye (DiOC6(3)), absolute counts of erythrocytes, granulocytes, and monocytes were obtained by means of flow cytometry (FC). The FC method has the potential for application to avian blood cells count; however, the method was unable to distinguish between lymphocytes and thrombocytes. In the present study, we improved the FC method to obtain separate counts of lymphocytes using DiOC5(3). After quail blood cells were stained with DiOC5(3), the cells were measured with FC. Each blood cell type was distinguished by means of their typical FL-1 (green fluorescence) and SSC (side scatter). Absolute numbers of erythrocytes, granulocytes, monocytes and lymphocytes in whole blood were obtained. The improved FC analysis worked equally well with chicken (Gallus gallus) and goose (Anser cygnoides) blood.

Topics: Animals; Carbocyanines; Flow Cytometry; Fluorescent Dyes; Lymphocyte Count; Lymphocytes; Quail

The toxicity and the genotoxicity of antimalarial alkaloid rich extracts derived from two plants used in traditional medicine in Mali (Mitragyna inermis (Willd.) O. Kuntze Rubiaceae and Nauclea latifolia (Sm.) Rubiaceae) were evaluated on in vitro and in vivo systems. The results demonstrated that an alkaloid rich extract derived from M. inermis induced a strong inhibition of protein synthesis in mammalian cells but did not exhibit mutagenic or genotoxic activity. An alkaloid rich extract derived from N. latifolia could interact in vitro with DNA of bacteria and mammalian cells, leading to G2-M cell cycle arrest and heritable DNA-damage, as well as inducing in vivo single-strand breaks in liver, kidney and blood cells.

Topics: Alkaloids; Animals; Antimalarials; Carbocyanines; Comet Assay; DNA Damage; Flow Cytometry; Humans; Kidney; Kinetics; Liver; Lymphocytes; Mali; Medicine, African Traditional; Membrane Potentials; Mice; Microscopy, Fluorescence; Monocytes; Mutagenicity Tests; Plant Extracts; Plants, Medicinal; Salmonella typhimurium

Hyperthermia induces cell death and the usual endpoint to study this is the ability of the cells to form colonies. Hyperthermia is also known to alter membrane characteristics, especially transmembrane potential and this has been correlated with duration and degree of heating. The aim of the present study was to see the correlation between changes in membrane potential and clonogenic ability of HeLa cells after heat treatment of 41-44 degrees C. Membrane potential was measured by the fluorescence polarization of the plasma membrane probe 3,3'-dipentyloxacarbocyanine by flow cytometry. Cell survival was assessed by colony formation assay. The fluorescence intensity increased and cell survival decreased with an increase in temperature. The fall in survival following heat treatment closely paralleled the increase in fluorescent intensity, especially heat treatments of 60 min or more. After 2 h of heating at 44 degrees C, the surviving fraction decreased to 1% and the fluorescence intensity increased to 154.84% of the unheated controls. This study suggests that measurement of membrane potential by flow cytometry may potentially be an alternative to colony forming assay for assessing cell survival. Since the results of membrane potential measurements are available immediately, this has implications for its potential use as a predictive assay of thermosensitivity.

Topics: Carbocyanines; Cell Membrane; Cell Survival; Clone Cells; Flow Cytometry; Fluorescent Dyes; HeLa Cells; Humans; Hyperthermia, Induced; Membrane Potentials

A flow cytofluorometric susceptibility test (FCST) was used for rapid determination of the susceptibility of Candida lusitaniae isolates to amphotericin B. The test is based on the decrease in fluorescence intensity of cells stained with 3,3'-dipentyloxacarbocyanine iodide (DiOC5(3)), a membrane potential-sensitive cationic dye, after drug treatment. A total of 58 C. lusitaniae clinical isolates including strains known to be amphotericin B-resistant on the basis of in-vivo and/or in-vitro data were tested. MICs were determined concurrently by the NCCLS broth macrodilution method and the Etest, both with antibiotic medium 3. Regression analysis demonstrated that the data from the FCST and the Etest were better correlated (r = 0.93, n = 59, P < 0.001) than those from the FCST and the NCCLS method (r = 0.63, n = 59, P < 0.001). The FCST readily identified a series of putatively susceptible and resistant isolates. Our study points out the advantages of the flow cytometry approach in antifungal susceptibility testing of yeasts, since speed remains a major problem in conventional tests.

Topics: Amphotericin B; Antifungal Agents; Candida; Carbocyanines; Drug Resistance, Microbial; Flow Cytometry; Fluorescent Dyes; Microbial Sensitivity Tests; Regression Analysis

Flow cytometry was used for measuring the effects of amphotericin B on the membrane of Leishmania infantum strains. The technique was adapted from the rapid flow cytometric membrane potential assay developed by Ordonez and Wehman (Cytometry 22:154-157, 1995) for evaluating antibiotic-susceptibility of Candida species. The study consisted of measuring membrane potential changes induced by amphotericin B in 3 initial strains and 12 laboratory-generated variants adapted to grow with amphotericin B. Results showed that, after 3 h of incubation, amphotericin B induced a dose-related decrease of membrane potential that reached its maximal level at the same concentrations that inhibited parasite growth. These results suggest that the flow cytometric membrane potential assay could be used to assess the susceptibility of Leishmania promastigotes to amphotericin B.

Topics: Amphotericin B; Animals; Antiprotozoal Agents; Carbocyanines; Dose-Response Relationship, Drug; Flow Cytometry; Fluorescent Dyes; Leishmania infantum; Membrane Potentials; Time Factors

The capacity of flow-cytometric techniques to detect drug-specific biochemical targets and side effects in Leishmania infantum promastigotes was estimated by assessing the effects of three antileishmanial drugs (pentamidine, allopurinol, and amphotericin B) on parasite metabolism. Cell cycle and total protein content were estimated by staining cells with propidium iodide and fluorescein isothiocyanate, nonprotein thiols were stained by mercury orange, and membrane potential was measured by the accumulation of 3,3'-dipenthyloxacarbocyanine iodide inside the cell. Results showed that dynamic studies in parasites treated with subtoxic concentrations of drugs allowed the detection of drug-specific targets: pentamidine primarily affected nonprotein thiol contents and DNA synthesis, allopurinol primarily affected intracellular protein contents, and amphotericin B primarily affected membrane potential. Moreover, the assessment of cellular functions in parasites treated with increasing concentrations of drugs certified the capacity of these techniques to establish dose-response curves and to permit the detection of side effects.

Topics: Allopurinol; Amphotericin B; Animals; Antiprotozoal Agents; Carbocyanines; Cell Cycle; Coloring Agents; DNA, Protozoan; Dose-Response Relationship, Drug; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Leishmania infantum; Membrane Potentials; Pentamidine; Phenylmercury Compounds; Propidium; Protozoan Proteins; Sulfhydryl Compounds; Sulfhydryl Reagents

Conventional techniques for antibiotic susceptibility testing usually require 24 h or more to produce accurate results. This long waiting period to demonstrate antibiotic action is necessary because such tests rely on growth (or the lack of it), when the microorganisms are incubated in the presence of the antibiotic. In an effort to improve antibiotic susceptibility testing, we developed a flow cytometric technique for Staphylococcus aureus in which antibiotic action is determined by monitoring drug-induced membrane potential changes, using the potential-sensitive dye 3,3'-dipentyloxacarbocyanine iodide. Three ATCC reference strains of S. aureus and 13 unknown strains of the same microorganism were tested for susceptibility to penicillin G and oxacillin. Our results indicate that susceptibility of S. aureus to these antibiotics can be measured reliably at 90 min after addition of the antibiotic, and the results are comparable to those obtained with conventional susceptibility tests.

Topics: Carbocyanines; Flow Cytometry; Fluorescent Dyes; Membrane Potentials; Microbial Sensitivity Tests; Oxacillin; Penicillin G; Staphylococcus aureus; Time Factors

In this study, we determined that three structurally related oxacarbocyanine dyes, 3,3'-diethyloxacarbocyanine (DiOC2(3)), 3,3'-dipentyloxacarbocyanine (DiOC5(3)), and 3,3'-dihexyloxacarbocyanine (DiOC6(3)), and one oxadicarbocyanine, 3,3'-diethyloxadicarbocyanine (DiOC2(4)), inhibit bovine heart mitochondrial NADH oxidase activity and one of them, DiOC6(3), inhibits Paracoccus denitrificans NADH oxidase activity. The mitochondrial I50 values were 9 microM (DiOC2(3)), approximately 1 microM (DiOC5(3)) and DiOC6(3)), and approximately 3 microM (DiOC2(4)), whereas the I50 value for P. denitrificans was approximately 2 microM (DiOC6(3)). Neither succinate nor cytochrome oxidase (EC 1.9.3.1) activity was inhibited significantly by any of the compounds in either electron transport chain, localizing the inhibitory site of the oxacarbocyanine dyes to the respiratory chain segment between NADH and ubiquinone. With submitochondrial particles (SMP), NADH-dependent reduction of duroquinone and coenzyme Q1 was inhibited markedly by all four compounds with DiOC6(3) being the most potent inhibitor, and the reduction of menadione was inhibited substantially by DiOC6(3). When purified complex I was used, NADH-dependent reduction of ferricyanide was inhibited by DiOC5(3) and coenzyme Q1 reduction was inhibited by all oxacarbocyanines. With P. denitrificans membrane vesicles, DiOC6(3) substantially inhibited NADH-dependent reduction of coenzyme Q1. All the oxacarbocyanines were more effective inhibitors with membrane preparations than with complex I, suggesting that membrane interactions play a role in inhibition. The mechanism of inhibition of the oxacarbocyanines appears to be similar to that of rotenone since (a) essentially only electron acceptors affected by rotenone were affected by the compounds, (b) inhibition of menadione reduction was diminished drastically with rotenone-saturated SMP, and (c) inhibition of coenzyme Q1 was largely eliminated with rotenone-insensitive complex I, and P. denitrificans membrane vesicles.

Topics: Animals; Carbocyanines; Cattle; Coloring Agents; Electron Transport; Electron Transport Complex I; Kinetics; Mitochondria, Heart; NAD; NADH, NADPH Oxidoreductases; Oxidation-Reduction; Oxidoreductases; Paracoccus denitrificans; Stimulation, Chemical; Structure-Activity Relationship

The effect of elevated temperature on transmembrane potential was studied in Chinese hamster ovary cells in vitro using tetraphenylphosphonium cation (TPP+) and 3,3'-dipentyloxacarbocyanine [Di-O-C5(3)], two unrelated lipophilic cation probes that equilibrate across the plasma membrane according to the transmembrane potential. Uptake of TPP+ was measured using a tritium-labeled probe and the uptake of the fluorescent probe Di-O-C5(3) was measured by flow cytometry. The Nernst equation was used to calculate transmembrane potential. The absolute values obtained for transmembrane potential at 37 degrees C using the two probes were different, but qualitatively similar results were obtained using either probe in the hyperthermia studies. Transmembrane potential measured at 43 and 45 degrees C was at least 20% higher than that measured at 37 degrees C, and the difference was statistically significant (P = 0.025 and P less than 0.01, respectively). The hyperpolarization induced by exposure to 45 degrees C persisted temporarily after cells had been returned to 37 degrees C. The hyperpolarization at 37 degrees C associated with a previous exposure to hyperthermia was maximal after cells had been held at 45 degrees C for 2.0 min, and fell to normal levels after 15.0 min at 37 degrees C.

Topics: Animals; Carbocyanines; Hot Temperature; Indicators and Reagents; Membrane Potentials; Onium Compounds; Organophosphorus Compounds

The chemotactic peptide formylmethionyl-leucyl-phenylalanine (fMLP), at concentrations below 10(-9) M, elicits a sustained increase in the human neutrophil's membrane potential within 10 s of its addition. This hyperpolarization, detected with the fluorescent cationic potentiometric probes, 3,3'-dipentyloxacarbocyanine (diO-C5-(3)), and 1,1'-dipropyl-3,3,3',3'-tetramethylindocarbocyanine iodide (diI-C3-(3)), and with the anionic probe bis-(1,3-diethylthiobarbituric)trimethine oxonol (bis-oxonol), is immediately followed by a large depolarization when [fMLP] greater than 10(-9) M. By extracellular substitution of sodium ions with potassium ions or choline or by pretreatment of the cells with ionophores, we report here that the hyperpolarization is primarily dependent on an intact potassium ion gradient and is accompanied by a concurrent acidification of the cytoplasm (approximately 0.05 pH unit) Although the latter occurs simultaneously with a large, transient increase in cytosolic Ca2+ at [fMLP] greater than 10(-10) M, it occurs without a detectable increase in cytosolic Ca2+ at [fMLP] less than 10(-10) M. The hyperpolarization is neither affected nor initiated by the chemotactic peptide antagonist tert-butyloxycarbonyl-methionyl-leucyl-phenylalanine, whereas the depolarization is completely inhibited. Neutrophils isolated from patients with X-linked chronic granulomatous disease exhibit normal hyperpolarizations and cytosolic Ca2+ increases in response to chemotactic peptides but exhibit no depolarization or oxidative burst. The hyperpolarization appears earlier in the ontogeny of differentiating myeloid precursor cells than either the rise in cytosolic Ca2+ or the depolarization response. Together, these findings indicate that an increase in transmembrane potential is one of the earliest events in the neutrophil response to chemotactic peptides, coinciding temporally with increases in cytoplasmic Ca2+ and H+ concentrations but preceding detectable oxidative burst activity.

Topics: Amino Acid Sequence; Calcium; Carbocyanines; Cell Membrane; Choline; Cytosol; Dose-Response Relationship, Drug; Fluorescent Dyes; Granulomatous Disease, Chronic; Humans; Hydrogen-Ion Concentration; Membrane Potentials; Molecular Sequence Data; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oligopeptides; Potassium; Potassium Channels; Sodium; Sodium-Potassium-Exchanging ATPase; Thiobarbiturates

Previous studies suggested that a 26 kDa protein might play an important role in protein synthesis-independent thermotolerance development in CHO cells. To determine if this phenomenon was universal, four mammalian cell lines, viz., CHO, HA-1, murine Swiss 3T3, and human HeLa, were studied. Cells were heated at 42 degrees C, and the level of 26 kDa protein in the nucleus was measured, together with clonogenic survival and protein synthesis. The results demonstrated that 1) the 26-kDa protein was present in the four different cell lines, and 2) the level of the 26 kDa protein in their nuclei was decreased by 30-70% after heating at 42 degrees C for 1 hr. However, restoration of this protein occurred along with development of chronic thermotolerance. The protein synthesis inhibitor cycloheximide (10 micrograms/ml) neither inhibited the development of chronic thermotolerance nor affected the restoration of the 26 kDa protein in the nucleus. In fact, this drug protected cells from hyperthermic killing and heat-induced reduction of 26 kDa protein in the nucleus. Heat sensitizers, quercetin (0.1 mM), 3,3'-dipentyloxacarbocyanine iodide (DiOC5[3]: 5 micrograms/ml), and stepdown heating (45 degrees C-10 min----42 degrees C), potentiated hyperthermic killing and inhibited or delayed the restoration of the 26 kDa protein to the nucleus. These results support a correlated, perhaps causal relationship between the restoration of the 26 kDa protein and chronic thermotolerance development in four different mammalian cell lines.

Topics: Animals; Biological Transport; Carbocyanines; Cell Line; Cell Nucleus; Cell Survival; Cricetinae; Cricetulus; Cycloheximide; Female; Heat-Shock Proteins; HeLa Cells; Hot Temperature; Humans; Molecular Weight; Nuclear Proteins; Quercetin; Time Factors

Chinese hamster cells (line CHO) stained with either 9 microM Hoechst 33342 (HO) alone or in combination with the membrane potential fluorochrome DiO-C5-3 (DiO) were analyzed using uv laser powers between 25 and 500 mW and sorted for determination of survival by a colony formation assay. The combination of HO-DiO increased fluorescence twofold and provided coefficients of variation (CVs) as low as 3.0% under conditions where viability of cells, even at 500 mW excitation, was unaffected. HO-stained cells yielded CVs of about 8.5% and survivals of approximately 90% under similar analytical conditions. At laser powers of 25 mW, CV values for HO-DiO-stained populations were 4.0% compared to 9.4% for HO-stained cells. Results with another membrane potential dye, rhodamine 123 (R 123), in combination with HO showed no improvement compared to HO-stained cells. No preferential, cell cycle phase-specific killing was observed in either the HO- or HO-DiO-stained populations. CVs of human skin diploid fibroblasts stained with HO or with HO-DiO were comparable over the entire laser power range; however, percentage survival was slightly higher for the HO-DiO-stained populations when analyzed and sorted at the higher power (400-500 mW) range. Long-term cultures of sorted CHO-K1 subpopulations, differing in DNA ploidy, were established from HO-DiO-stained cells. Advantages of this new staining procedure include improved DNA content resolution (low CV values) and the potential use of less expensive FCM uv laser systems coupled with less perturbing excitation powers.

Topics: Animals; Benzimidazoles; Carbocyanines; Cell Cycle; Cell Division; Cell Line; Cell Membrane; Cell Separation; Cell Survival; Cells, Cultured; DNA; Fibroblasts; Flow Cytometry; Fluorescent Dyes; Humans; Quinolines; Staining and Labeling

Human peripheral blood B cells were separated from monocytes and T cells, depleted of null cells by an anti-Leu 9 rosetting technique, and fractionated on discontinuous Percoll gradients to yield a highly purified, small, dense B-cell population. These cells responded to F(ab')2 goat anti-mu at 10 and 100 micrograms/ml with membrane depolarization (measured by immunofluorescence with 3,3'-dipentyloxacarbocyanine dye) at 1 h, cell volume enlargement by 48 h, and modest thymidine incorporation by 72 h. They also responded to the 12-kd human B-cell growth factor of Maizel with membrane depolarization, but not with cell volume increase. F(ab')2 anti-mu and B-cell growth factor together induced greater depolarization than was seen with either alone, but there was no synergy. The cell volume increase seen with F(ab')2 anti-mu was not increased by B-cell growth factor. Comparison of data analysis methods showed that mean fluorescence intensity most readily detected significant depolarization. We conclude that in human B cells: (1) depolarization may be a "general response" to a variety of membrane stimuli, because F(ab')2 anti-mu and B-cell growth factor acting through different receptors both induce it, and (2) depolarization does not inevitably lead to cell volume increase.

Topics: Antigen-Antibody Reactions; B-Lymphocytes; Carbocyanines; Cell Separation; Flow Cytometry; Fluorescent Dyes; Humans; Immunoglobulin mu-Chains; In Vitro Techniques; Interleukin-4; Interleukins; Lymphocyte Activation; Membrane Potentials

We demonstrated previously that variables of macrophage activation are associated with the development and progression of the arthritic lesion in the model of adjuvant induced arthritis. This association was investigated further by assessing the ability of antiarthritic agents to modulate variables of macrophage activation in direct comparison to effects on the arthritic lesion. Whereas indomethacin effectively reduced hindpaw edema, it had no significant effect on Ia expression or on any measurement of activation. Prednisolone inhibited hindpaw edema and the production of interleukin-1 (IL-1) by splenic macrophages. Only methotrexate inhibited hindpaw edema and all variables of macrophage activation (PGE2 and IL-1 production, cyanine dye accumulation) as well as the influx of Ia positive macrophages into synovial tissue.

Topics: Animals; Anti-Inflammatory Agents; Arthritis; Arthritis, Experimental; Carbocyanines; Dinoprostone; Edema; Fluorescent Dyes; Foot; Hindlimb; Interleukin-1; Interleukin-2; Macrophages; Male; Methotrexate; Monocytes; Rats; Rats, Inbred Lew; Spleen

Topics: Adolescent; Adult; Biomarkers; Carbocyanines; Child; Child, Preschool; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Fluorescent Dyes; Humans; In Vitro Techniques; Lymphocyte Activation; Membrane Potentials; Reference Values; T-Lymphocytes

Fluorescent molecules are widely used to study quantitative cell properties, such as density of different antigenic markers or membrane responses to various stimuli. In most cases, studies are done on bulk cell populations with a spectrofluorimeter or at the single cell level with a cytofluorograph. However, only microspectrofluorimetric techniques allow continuous recording of dynamic events undergone by individual cells. The aim of the present report was twofold: first, to describe a methodology easily accessible to cell biologists that allows simultaneous manipulation of single cells and measurements of their fluorescence properties; and second, through this methodology to study quantitative aspects of cell structure and function such as binding of a fluorescein-labeled lectin, transfer of fluorescent molecules between labeled and unlabeled cells brought in close contact, or fluorescence response of individual cells stimulated after being loaded with a potential-sensitive dye. We conclude that the understanding of many aspects of cell structure and behavior requires that individual cells be studied under dynamic conditions and for prolonged periods of time.

Topics: Animals; Carbocyanines; Cell Communication; Cell Line; Concanavalin A; Cytological Techniques; Erythrocytes; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; Humans; Leukocytes; Macrophages; Mice; N-Formylmethionine Leucyl-Phenylalanine; Rats; Rats, Inbred Strains; Spectrometry, Fluorescence; Thiocyanates; Thymus Gland

We isolated myeloid precursors from human marrow and studied the effects of phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) upon transmembrane potentials and cytosolic calcium ([Ca2+]i) as the cells matured. Using a panel of fluorescent probes, we found that membrane depolarization induced by PMA and fMLP in granulocytes, and elevation in [Ca2+]i stimulated by fMLP, were absent in myeloblasts. When we induced differentiation with granulocyte-macrophage colony-stimulating factors, we found that both ionic responses appeared at approximately the promyelocyte stage. By using di-O-C5(3), we detected an initial phase of fMLP-induced hyperpolarization which appeared ontogenetically earlier than depolarization and which could be evoked in mature granulocytes with lower concentrations of the ligand. Hyperpolarization was partially dependent on extracellular Na+, was abrogated by increasing the external K+ concentration, and was accompanied by mild acidification of the cytoplasm. Bordetella pertussis toxin abolished both hyperpolarization and depolarization. Our findings indicate that shifts in [Ca2+]i and membrane potential changes in response to PMA and fMLP evolve as granulocytes mature. In addition, transmembrane ionic fluxes induced by fMLP appear to be more complex than previously considered, involving at least two separable phases of membrane potential change.

Topics: Aminoquinolines; Benzothiazoles; Bone Marrow Cells; Calcium; Carbocyanines; Cell Differentiation; Cell Membrane; Cytosol; Fluorescent Dyes; Granulocytes; Hematopoietic Stem Cells; Humans; Hydrogen-Ion Concentration; Interleukin-3; Membrane Potentials; N-Formylmethionine Leucyl-Phenylalanine; Pertussis Toxin; Potassium; Sodium; Tetradecanoylphorbol Acetate; Virulence Factors, Bordetella

The measurement of time correlated intracellular mitochondrial staining with 3,3'-dipentyloxacarbocyanine [Di-O-C5(3)] appeared of interest to define the optimal staining conditions. Mitochondrial staining of lymphocytes, monocytes and granulocytes results in different fluorescence signals, related to the numbers of mitochondria, that are present in the cells of these various cell types. Alterations of Di-O-C(5)3 staining in a distinct cell type are due to changes in the physiological or functional state of the mitochondria. It appeared that such alterations occur in cells, which are cultured in the presence of cytosine arabinoside. The effect of cytotoxic drugs upon the mitochondrial membrane potential may be relevance for the understanding of the mechanism of action, exerted by cytotoxic drugs upon cell biology.

Topics: Carbocyanines; Cell Line; Cytarabine; Flow Cytometry; Granulocytes; Hematopoietic Stem Cells; Humans; Intracellular Membranes; Kinetics; Lymphocytes; Membrane Potentials; Mitochondria; Monocytes; Quinolines; T-Lymphocytes

Defective phagocytic cell function may partially account for the morbidity and mortality associated with thermal injury. In experimental thermal injury in the rat, small circulating blood volumes increase the difficulty in obtaining significant data. Furthermore, purification and or elicitation procedures have the potential for altering the cell surface characteristics and/or the functional response of the cell in question. We have examined the circulating neutrophils and pulmonary alveolar macrophages of anesthetized rats following a 16-20% body surface area scald injury to the shaved back. The circulating neutrophils of thermally injured rats were examined by flow cytometry following stimulation with phorbol myristate acetate (PMA) (100 ng/ml) in terms of the change in fluorescence intensity of the potentiometric cyanine dye, dipentyloxocarboxyanine and the formation of the oxidized product of 2',7'-dichlorofluorescin diacetate-loaded cells. The alveolar macrophages were examined after stimulation with PMA (100 ng/ml) in terms of the change in fluorescence intensity of the potentiometric dye, dipropylthiodicarbocyanine and the generation of superoxide production, as assessed by the superoxide dismutase inhibitable reduction of cytochrome c. Both cells exhibited a profound inhibition of cell function 4 hours after the insult, with partial return toward control values at later time points. Furthermore, the plasma of thermally injured rats, 4 hours after the burn was inhibitory to normal rat neutrophils. Fluorescent compounds suggestive of in vivo lipid peroxidation were maximally detectable at this time point. Further research is needed to establish the role of these products in the induction of phagocytic cell dysfunction.

Topics: Animals; Burns; Calcimycin; Carbocyanines; Flow Cytometry; Fluoresceins; In Vitro Techniques; Leukocyte Count; Macrophages; Neutrophils; Oxygen; Phagocytes; Plasma; Rats; Rats, Inbred Strains; Superoxides; Tetradecanoylphorbol Acetate

Stimulated neutrophils show ionic fluxes that may function as "transducers" between stimuli and effector functions. Using fluorescent probes, patterns of membrane-associated calcium (chlortetracycline, CTC) and membrane potential (3-3'-dipentyloxacarbocyanine, di-O-C5 (3)) in single living human neutrophils were observed with a fluorescence microscope fitted with an image intensifier and photometer. Fluorescence changes were related to chemiluminescence. In unstimulated neutrophils, CTC and di-O-C5 (3) fluorescence was brightest in the perinuclear region. Di-O-C5 (3) fluorescence was also seen in mitochondria. Neutrophil stimulation with zymosan, phorbol myristate acetate (PMA) or calcium ionophore (A23187) resulted in loss of di-O-C5 (3) and CTC fluorescence and chemiluminescence proportional to the strength of the stimulus. Experiments demonstrated the independence of these processes. (1) Digitonin stimulation caused chemiluminescence and di-O-C5 (3) darkening without loss of CTC fluorescence. (2) Depolarization of neutrophils did not induce CTC darkening or chemiluminescence. (3) Calcium ionophore (A23187) stimulation of neutrophils in calcium-free medium resulted in normal di-O-C5 (3) and CTC darkening, but a blunted chemiluminescence peak. (4) Calcium ionophore (A23187) stimulated the loss of di-O-C5 (3) and CTC fluorescence from chronic granulomatous disease neutrophils, but did not trigger an oxidative burst. Although neutrophil depolarization, calcium release from membranes, and oxidative activity are linked, these processes can clearly be separated.

Topics: Animals; Calcimycin; Calcium; Carbocyanines; Cell Membrane; Chickens; Chlortetracycline; Digitonin; Egtazic Acid; Humans; Luminescent Measurements; Membrane Potentials; Microscopy, Fluorescence; Neutrophils; Oxytetracycline; Zymosan

In an attempt to determine the mechanism of the profound defect in chemotaxis observed in the polymorphonuclear leukocytes (PMN) of human neonates, we have examined membrane potential changes and alterations in free intracellular calcium following chemotactic factor stimulation. Following exposure to formyl-methionyl-leucyl-phenylalanine (FMLP), PMN from adult donors (11) showed a marked change in membrane potential (31%) as determined by fluorescence emission using the cyanine dye, 3-3-dipentyloxacarbocyanine [DiOC5(3)]. In marked contrast, FMLP-stimulated PMN from 10 human neonates failed to show any significant change in membrane potential (1-2%). Using the calcium-sensitive probe Quin 2/AM, FMLP induced an increase in fluorescence of up to 51% in adult PMN (10). In contrast, the change in intracellular free calcium induced in neonatal PMN was much less (32%; P less than 0.01). These results suggest that the profound defect in chemotactic responsiveness of PMN from human neonates may result from an inability of these cells to undergo changes in membrane potential following inflammatory mediator stimulation.

Topics: Adult; Aminoquinolines; Carbocyanines; Cell Membrane; Chemotaxis, Leukocyte; Fetal Blood; Fluorescent Dyes; Humans; Infant, Newborn; Membrane Potentials; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Verapamil

Previous reports have suggested that histamine modulates neutrophil chemotaxis, but this has not been observed by all laboratories. We have re-addressed this controversial point and demonstrate that histamine and H1- and H2-receptor-specific agonists cause limited inhibition of chemotaxis while stimulating chemokinesis. Furthermore, using the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe) as a stimulus, we demonstrate that histamine and H1/H2 agonists inhibit f-met-leu-phe-stimulated changes in membrane potential (monitored with the cyanine dye dipentyloxacarbocyanine), superoxide anion production (cytochrome c reduction), hydrogen peroxide formation (scopoletin fluorescence), and degranulation of granule contents (lysozyme and beta-glucuronidase) in a dose-dependent manner but have no effect on neutrophil functions stimulated by the secretagogues phorbol myristate acetate or A23187. All inhibitory effects of histamine and the H1/H2 agonists are reversed in a competitive manner by the H2 antagonist cimetidine. In addition, structure-activity studies using H1 and H2 receptor agonists and antagonists indicate that a single site with specificity for both H1 and H2 analogue structures modulates the various f-met-leu-phe-stimulated functions studied. Kinetic studies demonstrate that the inhibitory effects of histamine on neutrophil function are only observed when histamine is added before f-met-leu-phe and that inhibition occurs within 10 to 20 sec of histamine addition, does not persist after its removal, and is reversed by addition of cimetidine 10 to 20 sec before stimulation with f-met-leu-phe. Although the inhibitory effects of histamine are exerted early in the sequence of PMN activation by f-met-leu-phe, histamine does not affect the binding or internalization of f-met-leu-[3H]phe. The ability of histamine to modify the variety of neutrophil responses demonstrated in this report suggests an important and direct role for histamine in the regulation of inflammatory reactions in acute allergic settings or other disease states in which histamine release may occur.

Topics: Carbocyanines; Chemotaxis, Leukocyte; Cytoplasmic Granules; Dimaprit; Glucuronidase; Histamine; Histamine H1 Antagonists; Histamine H2 Antagonists; Humans; Hydrogen Peroxide; Membrane Potentials; Muramidase; Neutrophils; Oxygen Consumption; Receptors, Histamine H1; Receptors, Histamine H2; Superoxides; Thiourea

The disruptive effects of Tris buffer and sodium lauroyl sarcosinate (Sarkosyl) on the outer membrane (OM) of Pseudomonas cepacia were investigated with several fluorescent probes. Tris increased the permeability of the OM to 6-anilino-l-naphthalenesulphonic acid and 2-p-toluidinylnaphthalene-6-sulphonate. The degree of damage to the OM was enhanced when the pH was decreased 3-(N-morpholino)propanesulphonic acid buffer had a small but significant effect at acid pH, while citrate/phosphate buffer showed insignificant effects. Sarkosyl released 3,3'-dipentyloxacarbocyanine iodide (CC5) from CC5-labelled OM or whole cells and altered OM fluidity as studied by fluorescence polarization.

Topics: Anilino Naphthalenesulfonates; Carbocyanines; Cell Membrane; Cell Membrane Permeability; Detergents; Fluorescence Polarization; Fluorescent Dyes; Hydrogen-Ion Concentration; Membrane Fluidity; Naphthalenesulfonates; Pseudomonas; Sarcosine; Surface-Active Agents; Tromethamine

We have employed a series of permeant, nontoxic, fluorescent probes to detect changes in ionic conditions within the mitotic apparatus of living endosperm cells of Haemanthus during the transition from metaphase to anaphase. Fluorescence emission intensity measurements from the spindle for chlorotetracycline (CTC) decline before the onset of anaphase, indicating a reduction in the amount of membrane-associated Ca2+ and suggesting an efflux of Ca2+ from membrane compartments into the spindle. Subsequent to the onset of anaphase, we observe increases in fluorescence with both 8-anilino-1-naphthalene sulfonate (ANS) and 3,3'-dipentyl 2,2'-dioxacarbocyanine (diO-C5(3)), sensitive to cationic and anionic charges at membrane surfaces, respectively. The increases with ANS and diO-C5(3) suggest that redistributions of ions within the spindle accompany anaphase motion. During the metaphase/anaphase transition, spindle membrane content remains constant, as evidenced by unchanging fluorescence with the hydrophobic probe, N-phenyl-1-naphthylamine (NPN). Shifts in emission intensity from the nonspindle cytoplasm or from the spindle poles do not accompany the changes in fluorescence we observe in the spindle, suggesting that any ionic fluxes responsible for the changes in fluorescence are restricted to the spindle domain.

Topics: 1-Naphthylamine; Anaphase; Anilino Naphthalenesulfonates; Calcium; Carbocyanines; Chlortetracycline; Fluorescent Dyes; Intracellular Membranes; Ions; Metaphase; Mitosis; Seeds

Previous studies using membrane potential sensitive probes have provided evidence that chemotactic factors elicit membrane potential changes in normal human neutrophils (PMN). In addition to stimulation of PMN motility, chemotactic factors also stimulate degranulation and superoxide ion (O-2) generation and it has been suggested that alteration of membrane potential activates these events (Korchak, H. M., and G. Weissmann. 1978. Proc, Natl, Acad, Sci. U. S. A. 75: 3818--3822). To further define the inter-relationship of these functions, studies were done with two indirect probes of membrane potential, 3-3'-dipentyloxacarbocyanine and triphenylmethylphosphonium ion (TPMP+) using PMN from normal subjects, from patients with abnormal O-2 production (chronic granulomatous disease [CGD]), and from patients with defective degranulation and/or chemotaxis (Cheddiak-Higashi syndrome and patients with elevated immunoglobulin (Ig)E and recurrent staphylococcal infections). The stimuli used were the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) and the secretagogues ionophore A23187 and phorbol myristate acetate (PMA). The results obtained with 3-3'-dipentyloxacarbocyanine and TPMP+ were comparable. The apparent membrane potential changes elicited by f-Met-Leu-Phe and PMA in normal PMN were reduced or entirely absent in PMN obtained from patients with CGD but normal in PMN from other patients. PMN from patients with CGD had normal calculated resting membrane potentials and normal responses elicited by the potassium ionophore valinomycin. The responses to calcium ionophore A23187 were only slightly impaired. The abnormality of the elicited response of CGD cells of f-Met-Leu-Phe and PMA could not be attributed to the absence of O-2, hydroxyl radical, singlet oxygen, or hydrogen peroxide acting on the probes. Instead this abnormality appears to be associated with a dysfunction in the normal molecular mechanism(s) stimulated upon neutrophil activation. The data suggest chemoattractant alteration of membrane potential in normal PMN is related to activation of oxidative metabolism but the relationship to chemotaxis and degranulation remains to be established.

Topics: Adolescent; Adult; Calcimycin; Carbocyanines; Chediak-Higashi Syndrome; Chemotactic Factors; Chemotaxis, Leukocyte; Child; Child, Preschool; Female; Granulomatous Disease, Chronic; Humans; In Vitro Techniques; Male; Membrane Potentials; Neutrophils; Onium Compounds; Superoxides; Tetradecanoylphorbol Acetate; Tetraphenylborate; Trityl Compounds; Valinomycin

Changes in the fluorescence intensity of the dye 3-3' dipentyloxacarbocyanine were measured in suspensions of purified human peripheral blood polymorphonuclear leukocytes (PMNs) during exposure to the chemotactic factors N-formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe) and partially purified C5a. Incubation of PMNs with dye resulted in a stable fluorescence reflecting the resting membrane potential of the cell. Exposure of PMNs to dye did not affect stimulated chemotaxis or secretion. The mechanism of cell-associated dye fluorescence involved solvent effects from partitioning of the eye between the aqueous incubation medium and the cell and not dye aggregation, Chemotactically active concentrations of f-met-leu-phe (5 x 10(-9) M or greater) produced a biphasic response characterized as a decrease followed by an increase in fluorescence. No fluorescence response was seen in lysed PMNs, and no response was elicited by an inhibitor of f-met-leu-phe binding (carbobenzoxy-phenylalanyl-methionine). The ability of several other synthetic peptides to elicit a fluorescence response corresponded to their effectiveness as chemotactic agents. Although the first component of the response suggested a depolarization, it was not influenced by variation in the external concentration of sodium, potassium, chloride, or calcium, and could not be characterized as a membrane potential change. The second component of the response, which was inhibited by both Mg2+ (10 mM)-EGTA (10 mM) and high external potassium, was compatible with a membrane hyperpolarization. The data indicate that chemotactic factors produce changes in dye fluorescence which can, at least in part, be attributed to a hyperpolarizing membrane potential change occurring across the plasma membrane.

Topics: Albumins; Animals; Carbocyanines; Chemotactic Factors; Chemotaxis, Leukocyte; Egtazic Acid; Fluorescent Dyes; Humans; Magnesium; Membrane Potentials; N-Formylmethionine; N-Formylmethionine Leucyl-Phenylalanine; Neuraminidase; Neutrophils; Oligopeptides; Potassium; Quinolines; Rats; Valinomycin

Potassium and sodium cation permeabilities of skeletal sarcoplasmic reticulum vesicles were characterized by means of 3H-choline, 22Na+ and 86Rb+ isotope efflux and membrane potential measurements. Membrane potentials were generated by diluting K gluconate filled sarcoplasmic reticulum vesicles and liposomes into Tris or Na gluconate media, in the presence or absence of valinomycin, and were measured using the voltage-sensitive membrane probe 3,3'-dipentyl-2,2'-oxacarbocyanine. About 2/3 of the sarcoplasmic reticulum vesicles, designated Type I, were found to be permeable to Rb+, K+ and Na+. The remaining 1/3, Type II vesicles, were essentially impermeable to these ions. The two types of vesicles were impermeable to larger cations such as choline or Tris. Both were present in about the same ratio in fractions derived from different parts of the reticulum structure. Studies with cations of different size and shape suggested that in Type I vesicles permeation was restricted to molecules fitting through a pore with a cross-section of 4--5 A by 6 A or more. When vesicles were sonicated, vesicles permeable to K+ decreased more than those impermeable to K+. These data suggest the existence of K+, Na+ permeable channels which are probably randomly dispersed in the intact reticulum structure at an estimated density of 50 pores/micrometer2. The function of the channel may be to allow rapid K+ movement to counter Ca2+ fluxes during muscle contraction and relaxation.

Topics: Animals; Calcium; Carbocyanines; Choline; Fluorescent Dyes; Ion Channels; Membrane Potentials; Muscles; Osmotic Pressure; Potassium; Rabbits; Rubidium; Sarcoplasmic Reticulum; Sodium