carbocyanines and 3-3--dipropyloxadicarbocyanine

The fluorescent carbocyanine dyes dil and diO have an extensive history of use in cell biology, but their use as neuronal tracers is relatively recent. We found in 1985 that these molecules were excellent retrograde and anterograde tracers in the developing nervous system. We went on to show that these dyes were retained in neurons placed in culture, that they initially labelled the processes as well as the cell bodies of cultured neurons, and that they were seemingly non-toxic. We suggested that the major mechanism of translocation for these molecules was lateral diffusion in the membrane, rather than fast axonal transport. This suggestion was recently confirmed in a striking manner by Godement et al., when they showed that these dyes can be used to label axonal projections in fixed tissues. Labelling with carbocyanine dyes has already allowed several exciting advances in developmental neurobiology. In this article we review the properties of carbocyanine dyes and point out some of their uses and advantages.

Topics: Animals; Axonal Transport; Axons; Carbocyanines; Cell Membrane; Fluorescent Dyes; Microscopy, Fluorescence; Neurons; Quinolines

Understanding the molecular mechanisms of skeletal muscle regeneration is crucial to exploiting this pathway for use in tissue repair. Our data demonstrate that the MEF2A transcription factor plays an essential role in skeletal muscle regeneration in adult mice. Injured Mef2a knockout mice display widespread necrosis and impaired myofiber formation. MEF2A controls this process through its direct regulation of the largest known mammalian microRNA (miRNA) cluster, the Gtl2-Dio3 locus. A subset of the Gtl2-Dio3 miRNAs represses secreted Frizzled-related proteins (sFRPs), inhibitors of WNT signaling. Consistent with these data, Gtl2-Dio3-encoded miRNAs are downregulated in regenerating Mef2a knockout muscle, resulting in upregulated sFRP expression and attenuated WNT activity. Furthermore, myogenic differentiation in Mef2a-deficient myoblasts is rescued by overexpression of miR-410 and miR-433, two miRNAs in the Gtl2-Dio3 locus that repress sFRP2, or by treatment with recombinant WNT3A and WNT5A. Thus, miRNA-mediated modulation of WNT signaling by MEF2A is a requisite step for proper muscle regeneration, and represents an attractive pathway for enhancing regeneration of diseased muscle.

Topics: Animals; Carbocyanines; Cell Line; Cells, Cultured; Chlorocebus aethiops; COS Cells; Frizzled Receptors; Gene Knockdown Techniques; Humans; MEF2 Transcription Factors; Mice; Mice, Knockout; MicroRNAs; Muscle, Skeletal; Myogenic Regulatory Factors; Regeneration; RNA, Long Noncoding; Signal Transduction; Up-Regulation; Wnt Proteins

The output of the hippocampus is largely determined by interaction of the three excitatory pathways that impinge on CA1 pyramidal neurons. These synapses, formed by axons of: (1) CA3 pyramidal neurons; (2) neurons of the entorhinal cortex (EC); and (3) neighboring CA1 neurons, are all potentially plastic. Here, we take advantage of the accessibility of the organotypic slice preparation to identify the type of spines with which each of these pathways forms synapses, at different developmental stages. Recent reports have shown that morphology of dendritic spines is activity-dependent with large mushroom spines being thought to represent stronger synaptic connections than thin or stubby spines. Although in a wide range of preparations, mushroom spines represent only 15% of spines across the whole dendritic tree, we find that this proportion is highly pathway specific. Thus in organotypic slices, the axons of CA3 neurons form synapses with mushroom spines on CA1 neurons in approximately 50% of cases, whereas this spine type is rare (<10%) in either of the other two pathways. This high proportion of mushroom spines only occurs after spontaneous excitatory activity in the CA1 cells increases over the second week in vitro. Previous studies suggest that pathway specificity also occurs in vivo. In tissue fixed in vivo, it is the synapses of distal apical dendrites thought to be formed by axons originating in the EC that are richer in mushroom spines. Hence, contrary to previous suggestions, the proportion of mushroom spines is clearly not an intrinsic property of the pathway but rather a characteristic dependent on the environment. We suggest that this is most likely a result of the previous activity of the synapses. The fact that, despite the large differences in pathway specificity between preparations, the overall proportion of different spine types remains unchanged, suggests a strong influence of homeostasis across the network.

Topics: Animals; Animals, Newborn; Carbocyanines; Dendritic Spines; Electric Stimulation; Hippocampus; Imaging, Three-Dimensional; In Vitro Techniques; Male; Neurons; Patch-Clamp Techniques; Perforant Pathway; Rats; Synapses; Synaptic Transmission; Time Factors

The adult facial nerve contains the axons from two populations of efferent neurons. First, the branchiomotor efferent neurons that innervate the muscles of the second arch. These neurons project out of the hindbrain in the motor root and form the facial motor nuclei. Second, the preganglionic efferent neurons that innervate the submandibular and pterygopalatine ganglia. These neurons project from the hindbrain via the intermediate nerve and form the superior salivatory nucleus. The motor neurons of the facial nerve are known to originate within rhombomeres 4 and 5. In the kreisler mouse mutant there is a specific disruption of the hindbrain rhombomeres 5 and 6 appear to be absent. To investigate changes in the organization of the facial motor neurons in this mutant, we have used lipophilic dyes to trace the facial motor components both retrogradely and anterogradely. As expected, facial motor neurons are missing from rhombomere 5 in this mutant. In addition, the loss of these neurons correlates with the specific loss of the superior salivatory nucleus. In contrast, the branchiomeric neurons, that originate in rhombomere 4, appear to develop normally. This includes the caudal migration of their cell bodies forming the genu of the facial nerve. Our studies confirm that rhombomeres are critical to hindbrain development and that they are the fundamental unit at which motor neurons are specified.

Topics: Animals; Carbocyanines; Facial Nerve; Fluorescent Dyes; Mice; Mice, Mutant Strains; Motor Neurons

Three fluorescent probes, tetramethyl rhodamine ethyl ester (TMRE), 3,3'-dipropylthiacarbocyanine iodide (diS-C3(3)) and 3,3'-dipropyloxacarbocyanine iodide (diO-C3(3)), were tested for their suitability as fluorescent indicators of membrane potential in Saccharomyces cerevisiae in studies performed by flow cytometry. For all these dyes the intensity of fluorescence of stained cells increased with probe concentration in the range of 60-3000 nmol/L. The optimum staining period was 15-20 min for diS-C3(3). Depolarization of cells by increased extracellular potassium level and by valinomycin elicited with all probes a drop in fluorescence intensity. In some yeast batches this depolarization was accompanied by a separation of subpopulations with different fluorescence properties.

Topics: Artifacts; Benzothiazoles; Carbocyanines; Flow Cytometry; Fluorescence; Fluorescent Dyes; Fluorometry; Indicators and Reagents; Membrane Potentials; Organometallic Compounds; Pyridinium Compounds; Saccharomyces cerevisiae; Scattering, Radiation; Time Factors

Topics: Animals; Antigens, Bacterial; Antigens, Viral; Carbocyanines; Fluorescent Dyes; Immunity, Mucosal; Immunization; In Vitro Techniques; ISCOMs; Lactobacillus; Macrophages; Mice; Mice, Inbred BALB C; Peyer's Patches; Rabies virus

We have developed a model system in the rat to test the feasibility of recombinant protein expression by genetically modified peritoneal mesothelial cells following autologous peritoneal implantation. Rat primary peritoneal mesothelial cells, isolated from parietal peritoneum by enzymatic digestion, were stably transduced (using a Moloney murine leukemia virus (MoMLV)-derived retroviral vector, BAG, expressing the Escherichia coli lacZ gene) to mark the cells with a reporter protein (beta-galactosidase, beta-gal). Such transduced mesothelial cells, tagged with DiO, a fluorescent lipophilic dye used for long-term tracing of transplanted cells, were then reseeded on the denuded peritoneal surface of syngeneic recipients. DiO-labeled, BAG-transduced mesothelial cells were observed to repopulate the denuded areas and remain attached there for > 90 days. Moreover, these genetically modified mesothelial cells continued to express the reporter gene product in vivo (ie beta-gal activity was present for at least 1 month). Our results demonstrate the feasibility of ex vivo gene therapy using peritoneal mesothelial cells.

Topics: Animals; beta-Galactosidase; Carbocyanines; Cell Line; Cells, Cultured; Epithelium; Escherichia coli; Female; Fluorescent Dyes; Genes, Bacterial; Genetic Therapy; Genetic Vectors; Moloney murine leukemia virus; Peritoneum; Rats; Rats, Inbred F344; Recombinant Proteins; Transfection; Transplantation, Homologous

Topics: Animals; Carbocyanines; Cell Death; Cell Survival; Cells, Cultured; Evaluation Studies as Topic; Female; Fluorescent Dyes; In Vitro Techniques; Insulin; Insulin Secretion; Islets of Langerhans; Male; Microscopy, Confocal; Propidium; Rats; Rats, Inbred Strains

Lipid-coated microbubbles (LCM) administered intravenously (i.v.) to rats bearing brain tumor, specifically enhance tumor visualization by ultrasound [1]. In order to understand the basis for this observation, we have examined the interactions of LCM with glioblastoma (C6) and gliosarcoma (9L) tumor cells in vivo and in vitro. LCM and LCM labeled with the fluorescent lipophilic dye 3,3'-dioctadecyloxacarbocyanine perchlorate (diO) were administered to rats bearing brain tumor. LCM and diO-labeled LCM were found principally at the tumor site with no evidence of label in the surrounding normal brain tissue. Analysis of the tumor by confocal laser scanning microscopy revealed that labeled LCM were inside the tumor cells. Similar analysis of LCM interactions with C6 and 9L cells in culture showed that LCM first adsorb at the surface of the cells, and with time became localized inside the cells. Binding and internalization proceeded faster at 37 degrees C than at room temperature (RT). Staining of live cells with N-(3-((2,4-dinitrophenyl)amino)propyl)-N-(3-aminopropyl) methylamine dihydrochloride (DAMP), a dye that recognizes acidic compartments, showed that the majority of internalized LCM was associated with compartments containing DAMP. If the same uptake mechanism were operative in vivo, it would indicate that a portion of LCM bypasses the reticuloendothelial system and become endocytosed directly by tumor cells.

Topics: Animals; Brain Neoplasms; Carbocyanines; Craniotomy; Dinitrobenzenes; Endocytosis; Fluorescent Dyes; Glioma; Gliosarcoma; Liposomes; Microscopy, Confocal; Microscopy, Fluorescence; Microspheres; Neoplasm Transplantation; Organelles; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Staining and Labeling; Tumor Cells, Cultured

Confrontation cultures between glioma spheroids and brain cell aggregates are well established in glioma research, and the model reflects several similarities to the in vivo brain tumour invasive process. The lipid-binding fluorescent carbocyanine dyes DiO (3,3'-dioctadecyloxacarbocyanine perchlorate) and DiI (1,1'-dioctadecyl-3,3,3,'3,'-tetramethylinocarbocyanine perchlorate) are widely used in cell biology as tracers for studying cell movement. Mature brain cell aggregates grown from fetal rat brain cells, and spheroids initiated from two glioma cell lines (GaMg and D-54Mg) were stained with DiO and DiI, respectively. Penetration of DiI and DiO into the tumour spheroids and brain aggregates was studied by confocal laser scanning microscopy (CLSM). After 48 h of dye exposures, the tracers had almost completely penetrated the tumour spheroids and brain aggregates. Light-microscopic sections of the specimens indicated that the dye incorporation had little effect on cellular morphology. Cell migration from DiI stained D-54Mg and GaMg spheroids was similar to that observed from unstained spheroids. Growth was also unaffected after 48 h of DiI exposure. Gioma cell invasion was assessed by CLSM using co-cultures of DiI -stained spheroids and DiO-stained brain cell aggregates. Optical sections revealed a gradual decrease in remaining brain volume, indicating a progressive invasive process. Single tumour cells were identified deep within the brain aggregates. In addition normal brain cells were also identified in the tumour spheroids. It is concluded that vital staining can be used to identify both normal cells and tumour cells during tumour cell invasion in vitro. The method may provide the possibility for studying the kinetics of single normal and tumour cell movement in individual tumour/brain co-cultures.

Topics: Animals; Brain; Brain Neoplasms; Carbocyanines; Cell Aggregation; Cell Division; Cell Movement; Coculture Techniques; Fetus; Fluorescent Dyes; Glioma; Humans; Microscopy, Confocal; Neoplasm Invasiveness; Rats; Rats, Inbred Strains; Tumor Cells, Cultured

The development of reticulospinal projections to the lumbar spinal cord is studied by using a collagen co-culture system. Outgrowing reticulospinal fibers seem to grow out in a straightforward direction, without a specific preference for the lumbar or tail spinal cord. Carbocyanine tracers such as DiI, DiO and DiA are used to label the outgrowing fibers or their parent cell bodies. In double labeling studies contacts of outgrowing reticulospinal fibers with lumbar motoneurons are analyzed. The advances of a confocal laser scanning microscope for such studies are illustrated.

Topics: Animals; Carbocyanines; Culture Techniques; Pyridinium Compounds; Reticular Formation; Spinal Cord; Xenopus laevis

We report results obtained in the microphthalmic strain of mice 944. Heterozygotes appear normal, but they produce litters in which typically between 2 and 5 offspring exhibit microphthalmia. As a result these animals are blind. Our investigations using the fluorescent tracer DiI show that in microphthalmic mice there is a small compensation for the missing retinal input by terminals or axon collaterals originating in the somatosensory thalamus. These morphological findings agree with somatosensory responses recorded in the visual cortex (EEG recordings).

Topics: Animals; Axons; Carbocyanines; Electroencephalography; Evoked Potentials; Fluorescent Dyes; Heterozygote; Mice; Mice, Mutant Strains; Microphthalmos; Reference Values; Visual Pathways

The projection pattern of the ventral thalamic reticular nucleus onto the dorsal thalamus was studied in the lizard Gallotia galloti using in vitro horseradish peroxidase and fluorescent carbocyanine labelling techniques. Localized label deposits at three dorsoventrally spaced sites in the dorsal thalamus elicited retrograde transport into separate, though partly overlapping, medial, dorsolateral and ventrolateral sectors within an extended cytoarchitectonic complex which may be globally identifiable as the reticular nucleus. Neurons found in the dorsolateral and ventrolateral sectors mainly corresponded to the cell group named nucleus ventromedialis (or nucleus of the dorsal supraoptic decussation) in the literature, whereas neurons labelled in the medial sector corresponded to the so-called dorsal hypothalamic nucleus. Sparser cells appear labelled in the superficially placed nucleus suprapeduncularis. Thalamotelencephalic fibers arising from the injected dorsal thalamic nuclei also project to the corresponding retrogradely labeled sectors within the reticular nucleus. These findings reveal a rough topographic organization in the connections of the extended reticular nucleus complex with the whole dorsal thalamus. This supports the hypothesis of hodological homology between this ventral thalamic formation in Gallotia and the mammalian thalamic reticular nucleus.

Topics: Animals; Brain Mapping; Carbocyanines; Fluorescent Dyes; Horseradish Peroxidase; Lizards; Neural Pathways; Thalamic Nuclei; Thalamus

A new assay using flow cytometry has been established to assess natural killer (NK) lytic activity with common bench top instrumentation. This assay uses a cyanine membrane dye to stain live K562 target cells and an iodide nuclear dye to evaluate dead cells, and provides a method of reliably separating target and effector cell populations. Effector cells remain unstained (fluorescent negative) throughout the procedure. The damaged pre-labeled targets appear doubly stained as their membranes become permeable to the nuclear stain during incubation. Percent cytotoxicity of various effector:target cell ratios is discerned using flow cytometric analysis after a 2 h incubation in this new assay, as compared to 4 h with the 51Cr-release 'gold standard' assay for cell-mediated cytotoxicity. Comparisons of normal individuals tested in parallel with the fluorescent dyes and the 51Cr-release assay have shown direct correlations. This new two-color flow cytometric technique has proven to be uncomplicated and reproducible when used in the clinical setting.

Topics: Adult; Carbocyanines; Chromium Radioisotopes; Cytotoxicity Tests, Immunologic; Evaluation Studies as Topic; Flow Cytometry; Fluorescent Dyes; Humans; In Vitro Techniques; Killer Cells, Natural; Kinetics; Tumor Cells, Cultured

Acutely isolated slices of developing rat hippocampus have been used to study axon growth and synapse formation. Mossy fibers, which are the axons of dentate granule cells, were labeled in living brain slices by injection of a fluorescent membrane dye (DiI or DiO) into the dentate gyrus. Time-lapse observations were made in area CA3 at a time when mossy fibers are normally growing in and forming en passant synapses with pyramidal neurons. Single scan images were collected at 1-2 min intervals over a period of several hours using a scanning laser confocal microscope. At the tips of growing mossy fibers were highly motile growth cones with several filopodia and small lamellae. Labeled fibers typically extended at rates up to 15 microns/h, but occasionally individual axons abruptly stopped elongating and the leading growth cone became quiescent. In addition, dynamic filopodia-like structures were found to be associated with axonal varicosities proximal to the leading growth cone. We are currently pursuing methods to determine whether these motile activities correlate with synapse formation.

Topics: Animals; Animals, Newborn; Carbocyanines; Fluorescent Dyes; Hippocampus; In Vitro Techniques; Microscopy, Fluorescence; Nerve Fibers; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Synapses

Cytotoxic T lymphocytes (CTL) can be very effective mediators of tumor-specific immunity in vivo. Since little is known about the in vivo behaviour of cultured tumor-specific CTL, a fast and simple method has been developed utilizing a lipophilic carbocyanine, 1,1'-dioctadecyl 3,3,3',3'-tetramethylin-docarbocyanine perchlorate (DiI), for the in vivo detection of tumor-specific CTL clones in (tumor-bearing) mice. The two CTL clones used in this study are directed against human papillomavirus type 16- or human adenovirus type 5 early region 1 (Ad5E1)-transformed mouse embryo cells. Growth ability, cytotoxic capacity and tumor-eradicating potential remained unaltered when the CTL were labeled with this dye. Thus, in neither in vitro nor in vivo testing was the biological function of the CTL clones affected. The in vivo localization in the spleen of an adoptively transferred DiI-labeled Ad5E1-specific CTL clone is described. This adoptively transferred CTL clone was also detectable at the site of a subcutaneously growing human Ad5E1-induced tumor within 1 day after intravenous injection.

Topics: Adenovirus Infections, Human; Affinity Labels; Animals; Carbocyanines; Clone Cells; Cytotoxicity, Immunologic; Fluorescent Dyes; Immunotherapy, Adoptive; Lymphocytes, Tumor-Infiltrating; Mice; Mice, Inbred C57BL; Mice, Nude; Papillomaviridae; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured; Tumor Virus Infections

The development of the adult visual system of Drosophila requires the establishment of precise retinotopic connections between retinal photoreceptor cell axons and their synaptic partners in the optic lobe of the brain. To assess the role of axon-axon interactions in retinal axon guidance, we used genetic methods to disrupt the normal spatiotemporal order of retinal axon ingrowth. We examined retinal axon projections to the developing first optic ganglion, the lamina, in two mutants in which reduced numbers of ommatidia develop in the eye imaginal disk. We find that in the developing lamina of these mutants, sine oculis and Ellipse, retinal axons project to proper dorsoventral positions despite the absence of the usual array of neighboring retinal axons. In a second approach, we examined animals that were somatic mosaics for the mutation, glass. In glass- animals, retinal axons project aberrantly and the larval optic nerve is absent. We find that in the developing lamina of glass mosaic animals, wild-type retinal axons project to proper dorsoventral positions despite the misrouted projections of neighboring glass- retinal axons. In addition, wild-type retinal axons project normally in the absence of the larval optic nerve, indicating that the latter is not an essential pioneer for retinal axon navigation. Our observations support the proposal that axon fascicles can make at least some pathfinding decisions independently of other retinal axon fascicles. We suggest that positional guidance cues that might label axon pathways and target destinations contribute to retinotopic pattern formation in the Drosophila visual system.

Topics: Animals; Carbocyanines; Cell Differentiation; Coloring Agents; Drosophila; Immunohistochemistry; Larva; Mutation; Neural Pathways; Optic Lobe, Nonmammalian; Optic Nerve; Photoreceptor Cells; Retina

Male canaries revise their vocal repertoire every year. Early work indicated that the volume and neuron number of the song-control nucleus HVC (Higher Vocal Center) declined in late-summer/fall as birds added and deleted syllables from their repertoire, and increased in spring as the set of song syllables stabilized to a fixed number. Seasonal variation in serum testosterone levels suggested that these changes in brain and behavior were regulated by testosterone (T). However, although initial studies describing growth and regression of HVC used Nissl-staining to define its borders, recent experiments that have measured the distribution of identified populations of HVC cells (projection neurons, hormone target cells) suggest that there are no seasonal changes in HVC volume or neuron number. In order to clarify the role of T in the regulation of HVC morphology, we castrated male canaries, maintained them on short (fall-like) days, and treated them with either T, antisteroid drugs, or nothing. After 1 month of treatment, we used a double-labeling technique to characterize HVC projection neurons and androgen target cells. The results showed that hormonal manipulation influenced HVC volume, the density and size of HVC cells, and the absolute number and percentage of androgen target cells in HVC. Hormonal manipulation did not influence the absolute number of cells in HVC. Moreover, the distribution of projection neurons, androgen target cells, and the Nissl-defined borders of HVC were closely aligned in all experimental groups, indicating that exposure to T and/or its metabolites (estradiol and dihydrotestosterone) regulates the overall size of HVC by affecting the distributions of both projection neurons and androgen target cells. Analysis of double-labeling results suggests that T specifically influences both cell size and the ability to accumulate androgen among HVC neurons that project to the robust nucleus of the archistriatum (RA). The results of this study show that steroid hormones exert potent effects on HVC morphology in male canaries, but differences between our results and studies of seasonal males suggest there may be additional factors that can regulate HVC morphology.

Topics: Animals; Birds; Brain; Carbocyanines; Fluorescent Dyes; Histocytochemistry; Hormones; Male; Microscopy, Fluorescence; Neurons; Orchiectomy; Seasons; Testosterone; Vocalization, Animal

A myelin basic protein (MBP)-reactive TH cell line capable of inducing experimental allergic encephalomyelitis (EAE), and a MBP-reactive TH cell clone that does not cause EAE were labeled with a fluorescent vital dye, and transferred into naive syngeneic SJL/J mice. Animals were killed before the appearance of symptoms (3 and 4 days post-injection). Sections obtained from the spleen, spinal cord and brain of both groups of animals were examined by fluorescence microscopy to localize labeled TH cells. At all time points examined, the spleens of both groups contained innumerable labeled cells. The spinal cords and brains of animals that had received EAE-causing cells had a basal level of 20 labeled cells/cm2 at 3 days; this number increased rapidly to 150 cells/cm2 in the spinal cord at 4 days. Perivascular infiltrates and small foci of astrogliosis were already apparent in this group 3 days after injection. The spinal cords and brains of animals that had received the non-EAE-causing TH cells contained 50 labeled cells/cm2 at 3 days. The density of these transferred cells, as compared to that of the EAE-causing cells, suggested that they have an unaltered CNS-homing capability. However, by 4 days, the number of non-EAE-causing labeled cells had returned to near basal level. Our findings suggest that discrimination between disease and non-disease causing MBP-responsive TH cells occurs within the first 3 days following transfer, requires the presence in the CNS of a limited number of TH cells, and depends on yet unidentified TH cell factor(s).

Topics: Animals; Autoimmune Diseases; Carbocyanines; Cell Line; Central Nervous System; Encephalomyelitis, Autoimmune, Experimental; Fluorescent Dyes; Mice; Microscopy, Fluorescence; Myelin Basic Protein; Receptors, Lymphocyte Homing; Spleen; T-Lymphocytes

A fast and simple method for the in vivo/in situ detection of liposomes is described. Utilizing lipophilic carbocyanine dyes, DiI and DiO, yellow (or red) and green fluorescent liposomes can be visualised with routinely available filters. The main advantages of the method are (i) the vesicles can be labelled after they are formed and (ii) the label does not interfere with proteins on the surface of the liposomes. Labelled liposomes were found in macrophages of spleen and liver (of mice) within 30 min of intravenous administration. In the spleen, labelled liposomes localised preferentially in the marginal zone macrophages, as confirmed by double staining with FITC-Ficoll. These data correlate well with the fact that empty or haptenated liposomes are thymus-independent antigens, and that other thymus-independent antigens are also specifically taken up by marginal zone macrophages. The immunological role of these macrophages in the processing and presentation of antigen-bearing liposomes can now be studied in more detail. Administration of high doses (1-3 mg lipid) of labelled liposomes showed that uptake occurred preferentially, but not exclusively, by marginal zone macrophages. After the marginal zone macrophages had been 'saturated', the red pulp macrophages took up the liposomes. DiI and DiO have also been successfully used for labelling lymphocytes and bacteria for in vivo homing studies. The fact that liposomes can be labelled after they have been formed is an advantage for retrospective (i.e. liposomes already in use/storage) studies in e.g. targeting of drugs by liposomes.

Topics: Affinity Labels; Animals; Carbocyanines; Female; Fluorescent Antibody Technique; Fluorescent Dyes; Kinetics; Liposomes; Liver; Macrophages; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Spleen

We describe a method for labeling cultured endothelial cells (ECs) and smooth muscle cells (SMCs) by letting the cells grow for three days in culture medium containing a low concentration of the fluorescent carbocyanine dyes DiI and DiO. We show that good labeling can be obtained with considerably lower concentrations (2.5 micrograms/ml) than has previously been described. With optimal concentration the labeling is very strong and seems to label all membranous structures in the cells. It was possible to clearly distinguish differentially pre-labeled cells both in coculture and seeded on denaturated vascular grafts. The cells remain fluorescent for more than seven days and may be passaged with retained proliferative capability. We suggest that DiI/DiO-labeling using dye-containing medium may be used for several cell types and is applicable in tissue culture and in the detection of implanted cells in vivo.

Topics: Carbocyanines; Cell Division; Cells, Cultured; Endothelium, Vascular; Fluorescent Dyes; Humans; Muscle Development; Muscle, Smooth, Vascular; Saphenous Vein

Schistosomula of Schistosoma mansoni bind human low density lipoproteins (LDL) in a concentration-dependent and saturable manner. The bound LDL could provide phospholipids and sterol to the worm, which cannot synthesize sterol de novo and lacks acyl chain-modifying capability. Here we have used three phospholipid analogues to explore the effect of LDL binding on the parasite's outer tegumental membrane, i.e. the outer of the two membranes that cover its surface syncytium. Fluorescein phosphatidylethanolamine (Fl-PE) and rhodamine phosphatidylethanolamine (Rh-PE) bound to the parasite in a saturable manner and, as shown by fluorescence microscopy, were confined to the surface. Fl-PE fluorescence was completely quenched by Trypan Blue and Fl-PE was lost from the surface following single exponential decay kinetics (t1/2 = 12 h), further suggesting that the probe was confined to the outer membrane. 1,1'-Dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-C18(3); DiI) did not bind saturably and was seen in both the surface and the internal parasite membranes. Fluorescence photobleaching recovery was used to measure the lateral mobility of Fl-PE in the outer membrane. The lateral diffusion coefficient of Fl-PE was approximately 10(-7) cm2s-1. The fractional mobility of Fl-PE was 85% when measured using a laser beam of radius 1.8 microns and 45% using a beam of radius 4.3 microns. These measurements suggest that the outer membrane is composed of micron-scale liquid crystalline-phase lipid domains that lack significant amounts of transmembrane proteins. LDL binding to the parasite surface did not alter the lateral mobility of Fl-PE or the rate of loss of either Fl-PE or Rh-PE.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Carbocyanines; Diffusion; Fluorescent Dyes; Humans; Lipoproteins, LDL; Membrane Lipids; Phosphatidic Acids; Rhodamines; Schistosoma mansoni

In vitro restoration of adriamycin sensitivity in a resistant human breast tumor cell line was obtained by continuous exposure to nanomolar nontoxic valinomycin concentrations. Seven-day treatment with nanomolar valinomycin concentrations caused a slight increase of the signal of the cationic fluorescent cyanine probe DiOC5(3) but did not appreciably affect adriamycin incorporation in the cells. A marked increase of the DiOC5(3) signal was obtained in the presence of micromolar valinomycin concentrations, which were incompatible with the in vitro cellular growth.

Topics: Carbocyanines; Cell Division; Doxorubicin; Drug Resistance; Drug Synergism; Fluorescent Dyes; Humans; Tumor Cells, Cultured; Valinomycin

Topics: Animals; Benzimidazoles; Carbocyanines; Cell Line; Cell Survival; Cricetinae; Cricetulus; DNA; Female; Fibroblasts; Flow Cytometry; Fluorescent Dyes; Ovary; Staining and Labeling

The lipophilic carbocyanine fluorescent label DiI was injected in one eye of aldehyde-fixed embryonic or postnatal hamsters and the brains were examined using flat-mounts of the chiasm region, of the lateral surface of the brainstem, or of the midbrain tectum. Single axons could be discerned within the optic nerves and along the optic tract. Many fibers were tipped by growth cones, ending at various levels of the brainstem. Fine details of retinofugal axon morphology, including varicosities, branch-points and filopodial extensions on growth cones were visible in the flat-mounts. Such preparations allow a high-resolution view of labeled axons which course near the surface of the brain. It is possible, with this method, to simultaneously examine the morphogenesis of multiple collateral arbors on single fibers which project to more than one terminal zone.

Topics: Animals; Animals, Newborn; Axons; Brain Stem; Carbocyanines; Cricetinae; Fetus; Histological Techniques; Retina; Synaptic Transmission; Visual Pathways

Hypercholesterolemic rabbit beta-VLDL and human LDL are both internalized by mouse peritoneal macrophages by receptor-mediated endocytosis. However, only beta-VLDL (which binds to the cells with a much higher affinity than LDL) markedly stimulates acyl-CoA/cholesterol acyl transferase (ACAT) and induces foam cell formation in these cells. As an initial step to test whether the two lipoproteins might be targeted to different organelles (which might differ in their ability to deliver cholesterol to microsomal ACAT), we studied the endocytic pathways of beta-VLDL and LDL. Lipoproteins were labeled with the non-transferable fluorescent label, DiI. When the macrophages were incubated with DiI-LDL for 10 min at 37 degrees C, the fluorescence was concentrated near the center of the cell both in heavily labeled vesicles and in a diffuse pattern. The pattern with DiI-beta-VLDL was quite different: an array of bright vesicles throughout the cytoplasm was the predominant feature. Differences in distribution were seen as early as 2 min of incubation and persisted throughout a 10-min chase period. By using a procedure in which photobleaching of DiI fluorescence converts diaminobenzidine into an electron-dense marker, we were able to identify at the ultrastructural level vesicles containing electron-dense material in cells incubated with DiI-beta-VLDL. Human E2/E2 beta-VLDL (from a patient with familial dysbetalipoproteinemia), which has a binding affinity and ACAT-stimulatory potential similar to LDL, gave a pattern of fluorescence virtually identical to LDL. Pulse-chase studies with 125I-labeled and [3H]cholesteryl ester-labeled lipoproteins disclosed that both protein degradation and cholesteryl ester hydrolysis were markedly retarded in beta-VLDL compared with LDL. Thus, in mouse peritoneal macrophages, endocytosed beta-VLDL appears in a distinct set of widely-distributed vesicles not seen with LDL (or with E2-beta-VLDL) and, compared with LDL, has a markedly diminished rate of protein degradation and cholesteryl ester hydrolysis. The differential routing of LDL and beta-VLDL may provide a mechanism for differences in ACAT-stimulatory potential between the two lipoproteins.

Topics: Animals; Carbocyanines; Cholesterol; Endocytosis; Female; Fluorescent Dyes; Lipoproteins, LDL; Lipoproteins, VLDL; Macrophages; Male; Mice; Microscopy, Electron; Microscopy, Fluorescence; Organelles; Peritoneal Cavity; Sterol O-Acyltransferase; Time Factors; Video Recording

The fluorescence intensity of the dye 1,1'-dipropylox-adicarbocyanine (DiOC3-(5] has been measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potential (Vm) under different ionic conditions, after treatment with cation ionophores and after hypotonic cell swelling. Calibration is performed with gramicidin in Na+-free K-/choline-media, i.e., standard medium in which NaCl is replaced by KCl and cholineCl and where the sum of potassium and choline is kept constant at 155 mM. Calibration by the valinomycin "null point" procedure described by Laris et al. (Laris, P.C., Pershadsingh, A., Johnstone, R.M., 1976, Biochim, Biophys. Acta 436:475-488) is shown to be valid only in the presence of the Cl- -channel blocker indacrinone (MK196). Distribution of the lipophilic anion SCN- as an indirect estimation of the membrane potential is found not to be applicable for the fast changes in Vm reported in this paper. Incubation with DiOC3-(5) for 5 min is demonstrated to reduce the Cl permeability by 26 +/- 5% and the NO3- permeability by 15 +/- 2%, while no significant effect of the probe could be demonstrated on the K+ permeability. Values for Vm, corrected for the inhibitory effect of the dye on the anion conductance, are estimated at -61 +/- 1 mV in isotonic standard NaCl medium, -78 +/- 3 mV in isotonic Na+-free choline medium and -46 +/- 1 mV in isotonic NaNO3 medium. The cell membrane is depolarized by addition of the K+ channel inhibitor quinine and it is hyperpolarized when the cells are suspended in Na+-free choline medium, indicating that Vm is generated partly by potassium and partly by sodium diffusion. Ehrlich cells have previously been shown to be more permeable to nitrate than to chloride. Substituting NO3- for all cellular and extracellular Cl- leads to a depolarization of the membrane, demonstrating that Vm is also generated by the anions and that anions are above equilibrium. Taking the previously demonstrated single-file behavior of the K+ channels into consideration, the membrane conductances in Ehrlich cells are estimated at 10.4 microS/cm2 for K+, 3.0 microS/cm2 for Na+, 0.6 microS/cm2 for Cl- and 8.7 microS/cm2 for NO3-. Addition of the Ca2+-ionophore A23187 results in net loss of KCl and a hyperpolarization of the membrane, indicating that the K+ permeability exceeds the Cl- permeability also after the addition of A23187. The K+ and Cl- conductances in A23187-treated Ehrlich cells are estimated at 134 and

Topics: Animals; Calcimycin; Carbocyanines; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Chlorides; Electric Conductivity; Fluorescent Dyes; Gramicidin; Ion Channels; Membrane Potentials; Mice; Quinine; Thiocyanates; Valinomycin

Organelle-depleted human neutrophil cytoplasts were used to study N-formylmethionylleucylphenylalanine (fmet-leu-phe) receptor regulation and adaptation, aggregation, chemotaxis, and chemoattractant-elicited changes in shape, volume, and surface charge. Cytoplasts aggregated in response to the chemoattractant fmet-leu-phe, but the magnitude of the response was less than that in neutrophils. Unlike neutrophils, cytoplasts did not exhibit a second wave of aggregation with the addition of cytochalasin B and also failed to disaggregate. In chemotactic assays, cytoplasts migrated poorly into cellulose nitrate filters, and compared with intact neutrophils required a 100-fold greater concentration of fmet-leu-phe to elicit shape change. In contrast to neutrophils, cytoplasts did not decrease their surface charge in response to fmet-leu-phe and did not exhibit an increase in the binding of fmet-leu-[3H]phe after stimulation with phorbol myristate acetate. However, receptor adaptation and induced changes in membrane potential, as measured by using the membrane probe di-O-C5(3), were similar in cytoplasts and in neutrophils. The data suggest that the presence of functional intracellular organelles is required for normal aggregation and disaggregation, chemotaxis, and shape change induced by fmet-leu-phe, and also peptide receptor upregulation in response to secretagogues. The data show that peptide receptor adaptation occurs in the absence of secretory granules and is independent of receptor upregulation .

Topics: Carbocyanines; Cell Aggregation; Chemotaxis, Leukocyte; Cytoplasmic Granules; Electrophoresis; Humans; Kinetics; Membrane Potentials; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Organoids; Receptors, Cell Surface; Receptors, Formyl Peptide; Tetradecanoylphorbol Acetate

The interaction of thymocyte mitochondria with two types of dyes - potential indicators commonly used in lymphocyte studies, has been investigated. Ethylrhodamine at concentrations up to 16 microM does not influence the systems of oxidation and energy coupling in lymphocyte mitochondria. Carbocyanines-diS-C3-(5) and diO-C3-(5) inhibit oxygen uptake by the lymphocytes in the presence of glucose and pyruvate at the same low concentrations as does rotenone (40% inhibition occurs at 10 nM). DNP reduces the inhibition of respiration by carbocyanines but not by rotenone. The increase in the fluorescence of diS-C3-(5) and in the rate of oxygen uptake in the absence of diS-C3-(5) occurs at close concentrations of the uncoupler. This indicates that the changes in the fluorescence caused by FCCP reflect the membrane potential of lymphocyte mitochondria. The maintenance of the membrane potential in lymphocyte mitochondria in the presence of diS-C3-(5) provides evidence for the absence of the corresponding changes in mitochondrial ultrastructure after addition of 0.6 microM diS-C3-(5) which completely inhibits oxygen uptake.

Topics: Animals; Benzothiazoles; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Fluorescent Dyes; Intracellular Membranes; Lymphocytes; Membrane Potentials; Mitochondria; Oxygen Consumption; Quinolines; Rats; Rhodamines; Thymus Gland; Xanthenes

The experiments presented below compare the interaction of diO-C3-(5) and diS-C3-(5) with erythrocytes, erythrocyte ghosts and phospholipid vesicles derived from erythrocyte membranes. The results confirm earlier reports of diS-C3-(5) dimerization in the presence of hemoglobin and of dye aggregate formation in erythrocyte suspensions. DiO-C3-(5), on the other hand, binds to vesicles and ghosts freed of hemoglobin in a potential-dependent manner but without forming dye aggregates. The two dyes bind to the different preparations in similar proportions, but diS-C3-(5) is bound in amounts 3-40 times greater depending on the degree of polarization. The results show that mechanism other than binding to hemoglobin must occur in order to explain the potential-dependent binding of both dyes to ghosts and vesicles. A primary interaction must exist between the dye molecule and the lipid bilayer in a biological membrane, and this would be expected to occur in the presence of hemoglobin or other cytosolic components. DiO-C3-(5) is a better dye to use than diS-C3-(5) for mechanistic studies, in order to avoid problems associated with formation of complex aggregates of the latter dye, especially in hyperpolarized membrane suspensions.

Topics: Benzothiazoles; Carbocyanines; Erythrocyte Membrane; Erythrocytes; Fluorescence; Fluorescent Dyes; Humans; Lipid Bilayers; Membrane Potentials; Potassium; Quinolines; Spectrophotometry

1. The magnitude of the K+ gradient across the plasma membrane, which was in equilibrium with the membrane potential (E) of the tumour cells, was determined by the "null point" procedure of Hoffman & Laris (1974) [J. Physiol. (London) 239, 519--552] in which the fluorescence of the dye serves as an indicator of changes in the magnitude of E. 2. A mixture of oligomycin, 2,4-dinitrophenol and antimycin was used to stop the mitochondria from interfering with the fluorescence signal. Transport functions at the plasmalemma were maintained under these conditions in the presence of glucose. 3. Physiological circumstances were found in which incubation with glycine or with glucose changed the "null point" value of E within the range--20mV to--100mV. The fluorescence intensity at the "null point" was an approximately linear function of E over that range. The procedure enabled E to be inferred form the fluorescence intensity in circumstances where titration to the "null point" was not feasible. 4. The rapid depolarization caused by l-methionine or glycine was shown in this way to have a maximum amplitude of about 60mV. A mathematical model of this process was devised. 5. The electrogenic Na+ pump hyperpolarized the cells up to about --80mV when the cellular and extracellular concentrations of K+ were roughly equal. 6. The observations show that the factors generating the membrane potential represent a major source of energy available for the transport of amino acids in this system.

Topics: Amino Acids; Animals; Antimycin A; Carbocyanines; Dinitrophenols; Fluorescent Dyes; In Vitro Techniques; Membrane Potentials; Mice; Mitochondria; Models, Biological; Neoplasms, Experimental; Oligomycins; Oxygen Consumption; Potassium; Quinolines; Sodium; Spectrometry, Fluorescence

1. Pigeon erythrocytes, resealed lysed erythrocytes or liposomes derived from erythrocyte lipids were suspended in solutions containing up to 2 micrometer-3,3'-dipropyloxadicarbocyanine iodide. Gramicidin, valinomycin, nigericin or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, or combinations of these, were used to induce electrical diffusion potentials dependent on Na+, K+ or protons. In each instance hyperpolarization of the cell membrane lowered the fluorescence of the cell suspension, a process that was completed in about 1 min. Subsequent depolarization caused an increase in fluorescence. 2. Quenching of the fluorescence of the cell suspension appeared to be due to the reversible binding of the dye to the cells. Much larger amounts of dye were bound, both to the intact and to the resealed erythrocytes, than would be expected if partitioning of the dye cation followed the Nernst equation. The dependence of the binding on the extracellular dye concentration was studied in the presence and absence of valinomycin. The results were consistent with the suggestion of Sims, Waggoner, Wang & Hoffman [(1974) Biochemistry 13, 3315-3330] that the dye was bound at both membrane surfaces and that, at low dye concentrations, hyperpolarizing the cells promoted dye binding at the inner membrane surface. 3. The applications of the technique are limited by the circumstance that the direct effect of the electric field on the uptake of the dye into the cells is amplified by a binding process that may be affected by other physiological variables.

Topics: Animals; Carbocyanines; Columbidae; Erythrocyte Membrane; Erythrocytes; Fluorescence; Fluorescent Dyes; Gramicidin; Ionophores; Liposomes; Membrane Potentials; Models, Biological; Nigericin; Quinolines; Valinomycin