Xenobiotic transport across isolated brain microvessels studied by confocal microscopy.
AuthorsMiller DS,Nobmann SN,Gutmann H,Toeroek M,Drewe J,Fricker G
JournalMolecular pharmacology
PubMed ID11093774
Activation of methotrexate-alpha-alanine by carboxypeptidase A-monoclonal antibody conjugate.
AuthorsHaenseler E, Esswein A, Vitols KS, Montejano Y, Mueller BM, Reisfeld RA, Huennekens FM
JournalBiochemistry
PubMed ID1731945
'Carboxypeptidase A (CP-A) and monoclonal antibody KS1/4 directed against an antigen on human lung adenocarcinoma cells (UCLA-P3) were derivatized by treatment with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and N-succinimidyl 3-(2-pyridyldithio)propionate, respectively. The derivatized proteins were reacted to produce thioether-linked enzyme-antibody conjugates. Sequential HPLC size-exclusion and DEAE chromatography separated the conjugate preparation from unreacted ... More
Relationship of F-actin distribution to development of polar shape in human polymorphonuclear neutrophils.
AuthorsCoates TD, Watts RG, Hartman R, Howard TH
JournalJ Cell Biol
PubMed ID1577856
'Polymerization of actin has been associated with development of polar shape in human neutrophils (PMN). To examine the relation of filamentous actin (F-actin) distribution to shape change in PMN, we developed a method using computerized video image analysis and fluorescence microscopy to quantify distribution of F-actin in single cells. PMN ... More
A lifetime-based optical CO2 gas sensor with blue or red excitation and stokes or anti-stokes detection.
AuthorsSipior J, Bambot S, Romauld M, Carter GM, Lakowicz JR, Rao G
JournalAnal Biochem
PubMed ID7573952
'We describe the fabrication and characterization of an optical CO2 sensor based on the change in fluorescence lifetimes due to fluorescence resonance energy transfer from a pH-insensitive donor, sulforhodamine 101, to a pH-sensitive acceptor, either m-cresol purple or thymol blue, entrapped in an ethyl cellulose film. A phase transfer agent ... More
Direct monitoring of vesicular release and uptake in brain slices by multiphoton excitation of the styryl FM 1-43.
AuthorsWinterer J, Stanton PK, Müller W
JournalBiotechniques
PubMed ID16568823
'Fluorescence imaging using FM 1-43 and related styryl dyes has provided invaluable insights into presynaptic function of synapses in culture preparations, but has been limited in use for studying central synapses in vivo or in brain slices, because of excessive fluorescence background due to nonspecific membrane binding of dye. We ... More
Rapid detection of mutagen induced micronucleated erythrocytes by flow cytometry.
AuthorsHutter KJ, Stöhr M
JournalHistochemistry
PubMed ID7141888
'The micronucleus test is a cytogenetic method for the screening of mutagens and carcinogens which exhibit clastogenic mechanisms of action. After application of clastogenic agents chromosomal fragments or even whole chromosomes can remain as conspicuous structures (micronuclei) in a small fraction of anucleated polychromatic erythrocytes which can be visually scored ... More
Laser mediated release of dye from liposomes.
AuthorsVanderMeulen DL, Misra P, Michael J, Spears KG, Khoka M
JournalPhotochem Photobiol
PubMed ID1438567
'Liposomes made from phospholipids and containing sulforhodamine dye (1-50 mM) have been irradiated with nanosecond and picosecond laser pulses. Individual liposomes were locally heated by laser absorption of dye dimers during a single laser pulse, and heating was sufficient to release the liposome contents. The extent of dye release produced ... More
Prescreening of cervical smears using two-parameter flow cytometry. Cytologic identification of artifacts.
AuthorsGieseking F, Baisch H, Scholz KU, Stegner HE, Linden WA
JournalAnal Quant Cytol
PubMed ID7332158
'Individual properties of gynecologic specimens can produce artifacts in flow cytometric (FMC) measurements, possibly leading to false interpretations. An identification of such artifacts was undertaken by parallel FCM and microscopic investigations. One hundred fifty unselected cervical smears were measured by FCM using the fluorochromes 4''-6-diamidino-2-phenylindole (DAPI) for DNA and sulforhodamine ... More
Time-resolved total internal reflection fluorometry study on polarity at a liquid/liquid interface.
AuthorsIshizaka S, Kim HB, Kitamura N
JournalAnal Chem
PubMed ID11403281
'The polarity of a water/oil (oil: cyclohexane, carbon tetrachloride, toluene, chlorobenzene, o-dichlorobenzene, or 1,2-dichloroethane) interface was investigated by means of time-resolved total-internal-reflection (TIR) fluorescence spectroscopy of a polarity-sensitive probe: sulforhodamine B (SRB). In bulk solutions, the nonradiative decay rate constant of SRB increased with an increase in a solvent polarity ... More
Toxicity of organic fluorophores used in molecular imaging: literature review.
AuthorsAlford R, Simpson HM, Duberman J, Hill GC, Ogawa M, Regino C, Kobayashi H, Choyke PL,
JournalMol Imaging
PubMed ID20003892
'Fluorophores are potentially useful for in vivo cancer diagnosis. Using relatively inexpensive and portable equipment, optical imaging with fluorophores permits real-time detection of cancer. However, fluorophores can be toxic and must be investigated before they can be administered safely to patients. A review of published literature on the toxicity of ... More
Sulphorhodamine-labelled cells in the neonatal rat spinal cord following chemically induced locomotor activity in vitro.
AuthorsKjaerulff O, Barajon I, Kiehn O
JournalJ Physiol
PubMed ID7525942
'1. Sulphorhodamine 101, a fluorescent dye and newly identified activity marker, was used to localize potential spinal locomotor networks in the neonatal rat spinal cord. 2. Preparations of the spinal cord with one entire hindlimb attached or the spinal cord in isolation were kept in vitro. Spinal locomotor activity was ... More
Spectra and fluorescence lifetimes of lissamine rhodamine, tetramethylrhodamine isothiocyanate, texas red, and cyanine 3.18 fluorophores: influences of some environmental factors recorded with a confocal laser scanning microscope.
AuthorsBrismar H, Trepte O, Ulfhake B
JournalJ Histochem Cytochem
PubMed ID7608524
'We report on the spectra and fluorescence lifetimes of four commonly used fluorophores: lissamine rhodamine (LRSC); tetramethyl rhodamine isothiocyanate (TRITC); Texas Red; and cyanine 3.18 (Cy-3). Fluorescence lifetime recordings revealed that these spectrally overlapping fluorophores can be individually detected by their lifetimes, indicating that at least four fluorophores can be ... More
A two-parameter flow cytometry protocol for the detection and characterization of the clastogenic, cytostatic and cytotoxic activities of chemicals.
AuthorsMaier P, Schawalder HP
JournalMutat Res
PubMed ID2431307
'Cultured, freshly-isolated rat fibroblasts were exposed in vitro to vincristine sulphate (VC), amethopterin (AM), bleomycin (BL), benomyl (BE) and practolol (PR). Cells treated for 5 h were subjected 24 h later to a two-parameter (DNA/protein) flow cytometry analysis. The fluorochromes used were sulphorhodamin 101 and DAPI. From DNA and protein ... More
Wavelength-resolved fluorescence detection in capillary electrophoresis.
AuthorsTimperman AT, Khatib K, Sweedler JV
JournalAnal Chem
PubMed ID7864386
'A multichannel laser-induced fluorescence detector for capillary electrophoresis is described. The detection system combines yoctomole limits of detection with the simultaneous acquisition of entire fluorescence emission spectra. An Ar/Kr mixed-gas ion laser provides great flexibility in excitation wavelengths, and a holographic grating and charge-coupled device detector combination allows a 500-nm ... More
A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry.
AuthorsGong J, Traganos F, Darzynkiewicz Z
JournalAnal Biochem
PubMed ID8074286
'In cells undergoing apoptosis (programmed cell death), a fraction of nuclear DNA is fragmented to the size equivalent of DNA in mono- or oligonucleosomes. When such DNA is analyzed by agarose gel electrophoresis it generates the characteristic "ladder" pattern of discontinuous DNA fragments. Such a pattern of DNA degradation generally ... More
Polyanionic agents as inhibitors of phagosome-lysosome fusion in cultured macrophages: evolution of an alternative interpretation.
AuthorsGoren MB, Vatter AE, Fiscus J
JournalJ Leukoc Biol
PubMed ID3468191
'Various natural and synthetic substances classified as polyanionics have been implicated in antagonizing phagosome-lysosome fusion in cultured macrophages. The phenomenon has been judged by comparing the transfer of selected markers from secondary lysosomes to phagosomes in control and in "polyanion" cells. Our earlier studies showed that use of one of ... More
Preparation of cell nuclei from fresh tissues for high-quality DNA flow cytometry.
AuthorsCastro J, Heiden T, Wang N, Tribukait B
JournalCytometry
PubMed ID8243208
'An easy method for preparation of bare cell nuclei from fresh solid tissues for DNA flow cytometry is described. Pieces of up to 2 x 2 x 2 mm3 size from fresh tissues were fixed in formalin. After removal of formalin by washing with ethanol and rehydration with tap water, ... More
In vitro eye-blink reflex model: role of excitatory amino acids and labeling of network activity with sulforhodamine.
AuthorsKeifer J
JournalExp Brain Res
PubMed ID7908648
'Evidence is presented suggesting that the neural correlate of the eye-blink reflex can be evoked in an in vitro brainstem-cerebellum preparation from the turtle by using electrical rather than natural stimulation of cranial nerve inputs. Abducens nerve discharge is triggered by brief electrical stimulation of the ipsilateral trigeminal nerve. This ... More
A lifetime-based fluorescence resonance energy transfer sensor for ammonia.
AuthorsChang Q, Sipior J, Lakowicz JR, Rao G
JournalAnal Biochem
PubMed ID8600839
'A lifetime-based optical NH3 sensor based on the principle of fluorescence resonance energy transfer was developed. The sensor consisted of sulforhodamine 101 as the donor, bromocresol green as the acceptor, ethyl cellulose as the polymer support, and tributyl phosphate as the plasticizer. When the concentration of NH3 changed, it caused ... More
Multiple innervation of tonic endplates revealed by activity-dependent uptake of fluorescent probes.
AuthorsLichtman JW, Wilkinson RS, Rich MM
JournalNature
PubMed ID3982503
'During development of the vertebrate nervous system, there is a widespread reduction in the number of axons innervating target cells. This phenomenon, often called synapse elimination, has been particularly well studied at the neuromuscular junction of developing twitch muscle fibres: following a period of polyneuronal innervation, axonal branches are retracted, ... More
Fluorescent labeling of cell surfaces.
AuthorsEdidin M
JournalMethods Cell Biol
PubMed ID2464121
Different quiescence states of three culture cell lines detected by acridine orange staining of cellular RNA.
AuthorsBaisch H
JournalCytometry
PubMed ID2456898
'Three cell lines (CHO, L-929, and R1H) were investigated for their growth kinetics and the difference of exponential and quiescent state of monolayers in medium with and without serum (L-929). The noncycling populations of L-929 and R1H in medium with serum contained increased G1-phase percentages but also considerable proportions of ... More
Nanochannels on a fused-silica microchip and liquid properties investigation by time-resolved fluorescence measurements.
AuthorsHibara A, Saito T, Kim HB, Tokeshi M, Ooi T, Nakao M, Kitamori T
JournalAnal Chem
PubMed ID12510735
'We have fabricated nanometer-sized channels, demonstrated a technique for the introduction of liquid into the channels, and carried out time-resolved fluorescence measurements of aqueous solutions. In this study, 330-nm- and 850-nm-sized channels were fabricated on fused-silica substrates by fast atom beam etching and hydrofluoric acid bonding methods. A liquid introduction ... More
Visualization of synaptic activity in hippocampal slices with FM1-43 enabled by fluorescence quenching.
AuthorsPyle JL, Kavalali ET, Choi S, Tsien RW
JournalNeuron
PubMed ID10624944
'Fluorescence imaging of presynaptic uptake and release of styryl dyes such as FM1-43 has provided valuable insights into synaptic function. However, in studies of CNS neurons, the utility of these dyes has been severely limited by nonsynaptic background fluorescence. This has thwarted the use of FM dyes in systems more ... More
Developmental profile and properties of sulforhodamine 101--Labeled glial cells in acute brain slices of rat hippocampus.
AuthorsKafitz KW, Meier SD, Stephan J, Rose CR,
JournalJ Neurosci Methods
PubMed ID18187203
'The reliable identification of astrocytes for physiological measurements was always time-consuming and difficult. Recently, the fluorescent dye sulforhodamine 101 (SR101) was reported to label cortical glial cells in vivo [Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. ... More
Xenobiotic efflux pumps in isolated fish brain capillaries.
AuthorsMiller DS, Graeff C, Droulle L, Fricker S, Fricker G
JournalAm J Physiol Regul Integr Comp Physiol
PubMed ID11742838
'To identify specific transporters that drive xenobiotics from the central nervous system to blood, the accumulation of fluorescent drugs was studied in isolated capillaries from killifish and dogfish shark brain using confocal microscopy and quantitative image analysis. In killifish brain capillaries, luminal accumulation of fluorescent derivatives of cyclosporin A and ... More
Terminal degeneration and synaptic disassembly following receptor photoablation in the retina of the fly's compound eye.
AuthorsBrandstätter JH, Shaw SR, Meinertzhagen IA
JournalJ Neurosci
PubMed ID2066769
'A long-term objective of our studies on the first optic neuropil (or lamina) underlying the fly''s compound eye is to explore how afferent photoreceptor synapses disappear during normal adult experience. To increase the frequency of this loss and the chances for its detection artificially, we have examined in this study ... More
Diffuse distribution of sulforhodamine-labeled neurons during serotonin-evoked locomotion in the neonatal rat thoracolumbar spinal cord.
AuthorsCina C, Hochman S
JournalJ Comp Neurol
PubMed ID10880990
'The fluorescent dye sulforhodamine-101 undergoes synaptic activity-dependent endocytotic uptake and consequent retrograde transport in presynaptic neurons. We used sulforhodamine to identify thoracolumbar spinal premotor neurons (T11-L6) activated during serotonin (5-HT) -induced hindlimb locomotor-like activity in the in vitro neonatal rat spinal cord preparation. Sulforhodamine labeling required locomotor-like activity because few ... More
Excitation spectra and brightness optimization of two-photon excited probes.
'Two-photon probe excitation data are commonly presented as absorption cross section or molecular brightness (the detected fluorescence rate per molecule). We report two-photon molecular brightness spectra for a diverse set of organic and genetically encoded probes with an automated spectroscopic system based on fluorescence correlation spectroscopy. The two-photon action cross ... More
Sulforhodamine labeling of neural circuits engaged in motor pattern generation in the in vitro turtle brainstem-cerebellum.
AuthorsKeifer J, Vyas D, Houk JC
JournalJ Neurosci
PubMed ID1494952
'A fluorescent molecular probe was used in combination with a novel in vitro preparation to study spatial patterns of neural activity associated with motor pattern generation. The in vitro brainstem-cerebellum preparation takes advantage of the turtle's unusual resistance to anoxia to preserve the entire neural network that connects the cerebellum, ... More
Sp1 and NF-Y are necessary and sufficient for growth-dependent regulation of the hamster thymidine kinase promoter.
AuthorsSorensen P, Wintersberger E
JournalJ Biol Chem
PubMed ID10521489
'Thymidine kinase (TK) genes from different species are growth- and cell cycle-regulated in a very similar manner; still, the promoter regions of these genes show little homology to each other. It was previously shown that the murine TK gene is growth-regulated by Sp1 and E2F. Here we have characterized cis-regulatory ... More
Synchronous hyperactivity and intercellular calcium waves in astrocytes in Alzheimer mice.
'Although senile plaques focally disrupt neuronal health, the functional response of astrocytes to Alzheimer''s disease pathology is unknown. Using multiphoton fluorescence lifetime imaging microscopy in vivo, we quantitatively imaged astrocytic calcium homeostasis in a mouse model of Alzheimer''s disease. Resting calcium was globally elevated in the astrocytic network, but was ... More
Direct vesicular transport of MHC class II molecules from lysosomal structures to the cell surface.
AuthorsWubbolts R, Fernandez-Borja M, Oomen L, Verwoerd D, Janssen H, Calafat J, Tulp A, Dusseljee S, Neefjes J
JournalJ Cell Biol
PubMed ID8909537
'Newly synthesized MHC class II molecules are sorted to lysosomal structures where peptide loading can occur. Beyond this point in biosynthesis, no MHC class II molecules have been detected at locations other than the cell surface. We studied this step in intracellular transport by visualizing MHC class II molecules in ... More
Astrocytic calcium signaling: mechanism and implications for functional brain imaging.
AuthorsWang X, Takano T, Nedergaard M,
JournalMethods Mol Biol
PubMed ID18839089
'Astrocytes are electrically non-excitable cells that, on a slow time scale of seconds, integrate synaptic transmission by dynamic increases in cytosolic Ca2+. A number of groups have recently shown that astrocytic Ca2+ signaling regulates vascular tones and that astrocytes play a central role in functional hyperemia by Ca2+ -dependent release ... More
Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo.
AuthorsGaraschuk O, Milos RI, Konnerth A
JournalNat Protoc
PubMed ID17406260
'One of the challenges for modern neuroscience is to understand the rules of concerted neuronal function in vivo. This question can be addressed using noninvasive high-resolution imaging techniques like two-photon microscopy. This protocol describes a versatile approach for in vivo two-photon calcium imaging of neural networks, stained with membrane-permeant fluorescent-indicator ... More
Gradual loss of synaptic cartels precedes axon withdrawal at developing neuromuscular junctions.
AuthorsBalice-Gordon RJ, Chua CK, Nelson CC, Lichtman JW
JournalNeuron
PubMed ID8240805
'We have studied the spatial deployment of synapses arising from different axons that converge on the same developing neuromuscular junctions. Labeling the competing synaptic "cartels" with different dyes in mouse muscle showed that, perinatally, each axon adds similar terminal areas, whereas later, areas occupied by the competing cartels diverged by ... More
Mechanism of concentration quenching of a xanthene dye encapsulated in phospholipid vesicles.
AuthorsPlant AL
JournalPhotochem Photobiol
PubMed ID3786467
Phase fluorometric method for determination of standard lifetimes.
AuthorsThompson RB, Gratton E
JournalAnal Chem
PubMed ID3382018
Applications of dye-sensitized photoreactions in neurobiology.
AuthorsSpikes JD
JournalPhotochem Photobiol
PubMed ID1775530
Corrected emission spectra and quantum yields for a series of fluorescent compounds in the visible spectral region.
AuthorsVelapoldi RA, Tønnesen HH,
JournalJ Fluoresc
PubMed ID15617389
This communication describes the determination of corrected fluorescence emission spectra and fluorescence quantum yields for fluorescein, sulforhodamine 101 and other standard fluorescent dyes.
Astrocytes going live: advances and challenges.
AuthorsNimmerjahn A,
JournalJ Physiol
PubMed ID19204050
Astrocytes are one of the most numerous cell types in the CNS. They have emerged as sophisticated cells participating in a large and diverse variety of functions vital for normal brain development, adult physiology and pathology. Recent in vivo studies have provided exciting new insight into astrocyte physiology in the ... More
The growth cones of identified motoneurons in embryonic zebrafish select appropriate pathways in the absence of specific cellular interactions.
AuthorsEisen JS, Pike SH, Debu B
JournalNeuron
PubMed ID2624743
Developing motoneurons in zebrafish embryos follow a stereotyped sequence of axonal outgrowth and accurately project their axons to cell-specific target muscles. During axonal pathfinding, an identified motoneuron pioneers the peripheral motor pathway. Growth cones of later motoneurons interact with the pioneer via contact, coupling, and axonal fasciculation. In spite of ... More
The Heidelberg flow analyzer and sorter (HEIFAS) approach on the prescreening of uterine cancer.
AuthorsStöhr M, Goerttler K
JournalJ Histochem Cytochem
PubMed ID86578
A dual laser flow system has been proved and used with a novel staining method for simultaneous quantitative DNA and protein analysis. A diamidinophenylindole compound (DAPI) for DNA has been employed in combination with sulforhodamine (SR 101) for protein. With this dye mixture various cell types of the cervical smear ... More
Polarization of chemoattractant receptor signaling during neutrophil chemotaxis.
Morphologic polarity is necessary for chemotaxis of mammalian cells. As a probe of intracellular signals responsible for this asymmetry, the pleckstrin homology domain of the AKT protein kinase (or protein kinase B), tagged with the green fluorescent protein (PHAKT-GFP), was expressed in neutrophils. Upon exposure of cells to chemoattractant, PHAKT-GFP ... More
Ischemia opens neuronal gap junction hemichannels.
AuthorsThompson RJ, Zhou N, MacVicar BA
JournalScience
PubMed ID16690868
Neuronal excitotoxicity during stroke is caused by activation of unidentified large-conductance channels, leading to swelling and calcium dysregulation. We show that ischemic-like conditions [O(2)/glucose deprivation (OGD)] open hemichannels, or half gap junctions, in neurons. Hemichannel opening was indicated by a large linear current and flux across the membrane of small ... More
Fluorescence detection in capillary zone electrophoresis using a charge-coupled device with time-delayed integration.
AuthorsSweedler JV, Shear JB, Fishman HA, Zare RN, Scheller RH
JournalAnal Chem
PubMed ID1905883
A fluorescence detection system for capillary zone electrophoresis is described in which a charged-coupled device (CCD) views a 2-cm section of an axially illuminated capillary column. The CCD is operated in two readout modes: a snapshot mode that acquires a series of images in wavelength and capillary position, and a ... More
Bipolar cells use kainate and AMPA receptors to filter visual information into separate channels.
AuthorsDeVries SH
JournalNeuron
PubMed ID11163271
Unlike cone photoreceptors, whose light responses have a uniform time course, retinal ganglion cells are tuned to respond to different temporal components in a changing visual scene. The signals in a mammalian cone flow to three to five morphologically distinct "OFF" bipolar cells at a sign-conserving, glutamatergic synapse. By recording ... More
Differential and sequential delivery of fluorescent lysosomal probes into phagosomes in mouse peritoneal macrophages.
AuthorsWang YL, Goren MB
JournalJ Cell Biol
PubMed ID2438290
It has previously been inferred that the fusion of a macrophage secondary lysosome with a phagosome delivers the entire lysosomal contents uniformly to the phagosome. We found, however, that different fluorescent lysosomal probes can enter phagosomes at remarkably different rates, even though they are initially sequestered together in the same ... More
'Photo-degeneration' of neurones after extracellular dye application.
AuthorsPicaud S, Wunderer HJ, Franceschini N
JournalNeurosci Lett
PubMed ID3226611
Irradiation in the presence of a dye applied to the extracellular space was found to trigger neuronal degeneration in fly photoreceptor neurones. This phenomenon endowed the selected cells with a dark and fine-grained label such that they could be traced and scrutinized for synaptic specializations. It also initiated their phagocytotic ... More
Directionally selective calcium signals in dendrites of starburst amacrine cells.
AuthorsEuler T, Detwiler PB, Denk W
JournalNature
PubMed ID12192402
The detection of image motion is fundamental to vision. In many species, unique classes of retinal ganglion cells selectively respond to visual stimuli that move in specific directions. It is not known which retinal cell first performs the neural computations that give rise to directional selectivity in the ganglion cell. ... More
Endocytic active zones: hot spots for endocytosis in vertebrate neuromuscular terminals.
AuthorsTeng H, Cole JC, Roberts RL, Wilkinson RS
JournalJ Neurosci
PubMed ID10366620
We have used a sensitive activity-dependent probe, sulforhodamine 101 (SR101), to view endocytic events within snake motor nerve terminals. After very brief neural stimulation at reduced temperature, SR101 is visualized exclusively at punctate sites located just inside the presynaptic membrane of each terminal bouton. The number of sites (approximately 26 ... More
From the Cover: Visually evoked activity in cortical cells imaged in freely moving animals.
We describe a miniaturized head-mounted multiphoton microscope and its use for recording Ca(2+) transients from the somata of layer 2/3 neurons in the visual cortex of awake, freely moving rats. Images contained up to 20 neurons and were stable enough to record continuously for >5 min per trial and 20 ... More
An identified motoneuron with variable fates in embryonic zebrafish.
AuthorsEisen JS, Pike SH, Romancier B
JournalJ Neurosci
PubMed ID2299397
Every trunk hemisegment of the zebrafish is innervated by 3 identified primary motoneurons whose development can be observed directly in living embryos. In this paper, we describe another identified neuron that is part of this system. Unlike the other primary motoneurons which are present in all trunk hemisegments, this cell ... More
Optical nanosensors for chemical analysis inside single living cells. 1. Fabrication, characterization, and methods for intracellular delivery of PEBBLE sensors.
AuthorsClark HA, Hoyer M, Philbert MA, Kopelman R
JournalAnal Chem
PubMed ID10565274
Spherical optical nanosensors, or PEBBLEs (probes encapsulated by biologically localized embedding), have been produced in sizes including 20 and 200 nm in diameter. These sensors are fabricated in a microemulsion and consist of fluorescent indicators entrapped in a polyacrylamide matrix. A generalized polymerization method has been developed that permits production ... More
Calcium imaging of living astrocytes in the mouse spinal cord following sensory stimulation.
AuthorsCirillo G, De Luca D, Papa M,
JournalNeural Plast
PubMed ID23091738
Astrocytic Ca(2+) dynamics have been extensively studied in ex vivo models; however, the recent development of two-photon microscopy and astrocyte-specific labeling has allowed the study of Ca(2+) signaling in living central nervous system. Ca(2+) waves in astrocytes have been described in cultured cells and slice preparations, but evidence for astrocytic ... More
Remote excitation of neuronal circuits using low-intensity, low-frequency ultrasound.
AuthorsTyler WJ, Tufail Y, Finsterwald M, Tauchmann ML, Olson EJ, Majestic C,
JournalPLoS ONE
PubMed ID18958151
Possessing the ability to noninvasively elicit brain circuit activity yields immense experimental and therapeutic power. Most currently employed neurostimulation methods rely on the somewhat invasive use of stimulating electrodes or photon-emitting devices. Due to its ability to noninvasively propagate through bone and other tissues in a focused manner, the implementation ... More
Synaptically induced sodium signals in hippocampal astrocytes in situ.
AuthorsLanger J, Rose CR,
JournalJ Physiol
PubMed ID19858225
Astrocytes are in close contact to excitatory synapses and express transporters which mediate the sodium-dependent uptake of glutamate. In cultured astrocytes, selective activation of glutamate transport results in sodium elevations which stimulate Na(+)/K(+)-ATPase and glucose uptake, indicating that synaptic release of glutamate might couple excitatory neuronal activity to glial sodium ... More
High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision.
Two-photon calcium imaging of neuronal populations enables optical recording of spiking activity in living animals, but standard laser scanners are too slow to accurately determine spike times. Here we report in vivo imaging in mouse neocortex with greatly improved temporal resolution using random-access scanning with acousto-optic deflectors. We obtained fluorescence ... More
Clathrin-mediated endocytosis in snake motor terminals is directly facilitated by intracellular Ca2+.
AuthorsTeng H, Wilkinson RS,
JournalJ Physiol
PubMed ID15860527
At the snake neuromuscular junction, low temperature (LT, 5-7 degrees C) blocks clathrin-mediated endocytosis (CME) while exocytosis is largely unaffected. Thus compensatory endocytosis that normally follows transmitter release is inhibited, or 'delayed' until the preparation is warmed to room temperature (RT). This delay was exploited to observe how changes in ... More
Radially expanding transglial calcium waves in the intact cerebellum.
Multicellular glial calcium waves may locally regulate neural activity or brain energetics. Here, we report a diffusion-driven astrocytic signal in the normal, intact brain that spans many astrocytic processes in a confined volume without fully encompassing any one cell. By using 2-photon microscopy in rodent cerebellar cortex labeled with fluorescent ... More
Assessment of golgi apparatus versus plasma membrane-localized multi-drug resistance-associated protein 1.
AuthorsKaufmann AM, Toro-Ramos AJ, Krise JP,
JournalMol Pharm
PubMed ID18557629
Traditionally, proteins belonging to the ATP-binding cassette superfamily have been thought to function exclusively at the plasma membrane (PM) of cells. We have previously shown multidrug resistance-associated protein 1 (MRP1) to reside on the Golgi apparatus of the multidrug resistant (MDR) human leukemic cell line HL-60 (HL-60/ADR); however, neither the ... More
Fluorescence spectroscopic profiling of compound libraries.
AuthorsSimeonov A, Jadhav A, Thomas CJ, Wang Y, Huang R, Southall NT, Shinn P, Smith J, Austin CP, Auld DS, Inglese J,
JournalJ Med Chem
PubMed ID18363325
Chromo/fluorophoric properties often accompany the heterocyclic scaffolds and impurities that comprise libraries used for high-throughput screening (HTS). These properties affect assay outputs obtained with optical detection, thus complicating analysis and leading to false positives and negatives. Here, we report the fluorescence profile of more than 70,000 samples across spectral regions ... More
Two-wavelength mercury arc lamp excitation for flow cytometric DNA-protein analyses.
AuthorsHeiden T, Göhde W, Tribukait B
JournalAnticancer Res
PubMed ID1704694
Correlated DNA- and protein-determinations were performed using flow cytometry and DAPI-sulforhodamine 101 (SR101) staining. Two-wavelength UV-green excitation using a single HBO lamp was found to be superior to single wavelength UV excitation, since it resulted in a higher specificity in the SR101 measurements. The effects of overlapping in the emission ... More
Selective chemical treatment of cellular microdomains using multiple laminar streams.
AuthorsTakayama S, Ostuni E, LeDuc P, Naruse K, Ingber DE, Whitesides GM
JournalChem Biol
PubMed ID12618184
There are many experiments in which it would be useful to treat a part of the surface or interior of a cell with a biochemical reagent. It is difficult, however, to achieve subcellular specificity, because small molecules diffuse distances equal to the extent of the cell in seconds. This paper ... More
Skin electroporation: rapid measurements of the transdermal voltage and flux of four fluorescent molecules show a transition to large fluxes near 50 V.
AuthorsChen T, Segall EM, Langer R, Weaver JC
JournalJ Pharm Sci
PubMed ID9811492
Large molecular fluxes and tight control are highly desired for transdermal drug delivery, which motivated this study of molecular transport due to high voltage pulsing. We used a flow-through sampling system (time resolution of approximately 14 s) to measure the response of human skin in vitro to a series of ... More
Development of a human nasal epithelial cell culture model and its suitability for transport and metabolism studies under in vitro conditions.
AuthorsWerner U, Kissel T
JournalPharm Res
PubMed ID7596993
A human nasal epithelial cell culture model has been adapted to observe transport and metabolism of drugs, e.g., peptides. Human nasal epithelial cells, isolated by protease treatment of human nasal conchae, grew to confluency after 6-8 days using DMEM supplemented with 1% nonessential amino acids, 1% glutamine, 10% FCS and ... More
Simultaneous measurement of DAPI-sulforhodamine 101 stained nuclear DNA and protein in higher plants by flow cytometry.
AuthorsUlrich W
JournalBiotech Histochem
PubMed ID1376151
A 2-step staining procedure is presented for simultaneous measurement of nuclear DNA and protein content in higher plants by flow cytometry. To release nuclei, plant tissues were chopped and stirred in the presence of the DNA specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI) and the nonionic detergent Triton X-100. Plant protoplasts were stirred ... More
Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo.
AuthorsNimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F
JournalNat Methods
PubMed ID15782150
Glial cells have been identified as key signaling components in the brain; however, methods to investigate their structure and function in vivo have been lacking. Here, we describe a new, highly selective approach for labeling astrocytes in intact rodent neocortex that allows in vivo imaging using two-photon microscopy. The red ... More
Flow cytometric applications of Sulforhodamine 101 as a fluorescent stain for total cellular protein.
AuthorsEngelhard HH
JournalBiotech Histochem
PubMed ID9062703
In this paper, the use of Sulforhodamine 101 (SR 101; C.I. 14318) as a fluorescent stain for flow cytometric determinations of total cellular protein (TCP) is described. Flow cytometric quantification of TCP fluorescence can provide a valuable analytical parameter for assessing both changes occurring in overall cellular protein content, such ... More
Modular transport of postsynaptic density-95 clusters and association with stable spine precursors during early development of cortical neurons.
AuthorsPrange O, Murphy TH
JournalJ Neurosci
PubMed ID11717366
The properties of filopodia and spines and their association with the postsynaptic density (PSD) protein PSD-95 were studied during early development of cultured cortical neurons using time-lapse confocal microscopy. Neurons were transfected with recombinant PSD-95 constructs fused to green fluorescent protein (GFP) for, on average, either 8 d in vitro ... More
Aneuploid and polyploid cellular DNA heterogeneity in insect cell material of diptera species analyzed by flow cytometry.
AuthorsUlrich W
JournalZ Naturforsch [C]
PubMed ID2127179
Flow cytometric 1-parameter DNA analysis and 2-parameter DNA/protein analysis have been performed with cell material of the diptera species Chironomus thummi, Drosophila melanogaster, Calliphora vicina and Musca domestica using an impulse cytophotometer with a new quartz objective, that was especially manufactured for cytofluorometric investigations. The occurrence of heterogenous cell populations ... More
Caffeine interaction with fluorescent calcium indicator dyes.
AuthorsMuschol M, Dasgupta BR, Salzberg BM
JournalBiophys J
PubMed ID10388782
We report that caffeine, in millimolar concentrations, interacts strongly with four common calcium indicator dyes: mag-fura-2, magnesium green, fura-2, and fluo-3. Fluorescence intensities are either noticeably enhanced (mag-fura-2, fura-2) or diminished (magnesium green, fluo-3). The caffeine-induced changes in the fluorescence spectra are clearly distinct from those of metal ion binding ... More
Dye-induced photopermeabilization and photodegeneration: a lesion technique useful for neuronal tracing.
AuthorsPicaud S, Wunderer H, Franceschini N
JournalJ Neurosci Methods
PubMed ID2232863
Using as a neural system the fly retina, which is visually accessible in vivo, we describe a lesion technique that takes advantage of the photodynamic damage produced by extrinsic dyes. Contrary to the photo-inactivation technique described by Miller and Selverston (1979), this technique does not involve intracellular injection, since the ... More
Regulation of MRP2-mediated transport in shark rectal salt gland tubules.
AuthorsMiller DS, Masereeuw R, Karnaky KJ
JournalAm J Physiol Regul Integr Comp Physiol
PubMed ID11832398
We examined endothelin-1 (ET-1) regulation of the xenobiotic efflux pump, multidrug resistance-associated protein isoform 2 (MRP2), in intact dogfish shark rectal salt gland tubules using a fluorescent substrate sulforhodamine 101 and confocal microscopy. Subnanomolar to nanomolar concentrations of ET-1 rapidly reduced the cell-to-lumen transport of sulforhodamine 101. These effects were ... More
Direct measurement of trans-Golgi pH in living cells and regulation by second messengers.
AuthorsSeksek O, Biwersi J, Verkman AS
JournalJ Biol Chem
PubMed ID7890600
In the endocytic compartment, an acidic pH plays a key role in receptor and ligand sorting, vesicular transport, and protein degradation. In the secretory compartment, indirect estimates of trans-Golgi pH based on partitioning of weak bases and following viral infection suggest a mildly acidic pH of > 6.0. We developed ... More
Evaluation of eight fluorochrome combinations for simultaneous DNA-protein flow analyses.
AuthorsStöhr M, Vogt-Schaden M, Knobloch M, Vogel R, Futterman G
JournalStain Technol
PubMed ID83691
Eight fluorescent dye combinations for simultaneous DNA-protein staining have been evaluated spectroscopically and flow microfluorometrically: propidium iodide (PI) with fluoresceinisothiocyanate (FITC), fluorescamine (FC), and dansylchloride (DANS); diamidinophenylindole (DAPII) with sulphorhodamin (SR101), tetramethylrhodamin isothiocyanate (TRITC), and nitrobenzodiazole (NBD); acriflavine (AF) with stilbene isothiocyanate sulphonic acid (SITS), and DAPI. Three different experimental ... More
Dynamic behavior of rod photoreceptor disks.
AuthorsChen C, Jiang Y, Koutalos Y
JournalBiophys J
PubMed ID12202366
Eukaryotic cells use membrane organelles, like the endoplasmic reticulum or the Golgi, to carry out different functions. Vertebrate rod photoreceptors use hundreds of membrane sacs (the disks) for the detection of light. We have used fluorescent tracers and single cell imaging to study the properties of rod photoreceptor disks. Labeling ... More
Excretory transport of xenobiotics by dogfish shark rectal gland tubules.
Marine elasmobranch rectal gland is a specialized, osmoregulatory organ composed of numerous blind-ended, branched tubules emptying into a central duct. To date, NaCl excretion has been its only described function. Here we use isolated rectal gland tubule fragments from dogfish shark (Squalus acanthias), fluorescent xenobiotics, and confocal microscopy to describe ... More
Primitive, crustacean-like state of blood-brain barrier in the eye of the apterygote insect Petrobius (Archaeognatha) determined from uptake of fluorescent tracers.
AuthorsShaw SR, Varney LP
JournalJ Neurobiol
PubMed ID10590170
Compound eyes of insects in 16 orders were tested for the presence of a blood-retina barrier (BRB) by injecting the hemolymph with Procion yellow, which was excluded from the eye in all Neoptera but not in two apterygotes. A primitive apterygote (Petrobius, Machilidae) was investigated further. Epifluorescence observations with small ... More
Simultaneous quantification of c-myc oncoprotein, total cellular protein, and DNA content using multiparameter flow cytometry.
AuthorsEngelhard HH, Krupka JL, Bauer KD
JournalCytometry
PubMed ID1900228
Variations in total cellular protein content can confound interpretation of the significance of modulations of specific cellular proteins. In an effort to overcome this problem, a technique is described for the simultaneous measurement of a specific cellular protein, total cellular protein, and DNA content. The method utilizes dual-laser (uv and ... More
Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis.
Authorsvan der Luit AH, Budde M, Ruurs P, Verheij M, van Blitterswijk WJ
JournalJ Biol Chem
PubMed ID12183451
The synthetic alkyl-lysophospholipid (ALP), 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, is an antitumor agent that acts on cell membranes and can induce apoptosis. We investigated how ALP is taken up by cells, how it affects de novo biosynthesis of phosphatidylcholine (PC), and how critical this is to initiate apoptosis. We compared an ALP-sensitive mouse lymphoma ... More
There is substantial nuclear and cellular disintegration before detectable phosphatidylserine exposure during the camptothecin-induced apoptosis of HL-60 cells.
AuthorsKing MA, Radicchi-Mastroianni MA, Wells JV
JournalCytometry
PubMed ID10754512
BACKGROUND: An early sign of apoptosis in many cells is the appearance of phosphatidylserine (PS) on the outside of the plasma membrane, whilst the cells still retain the ability to exclude DNA-binding molecules such as propidium iodide and 7-aminoactinomycin D (7-AAD). The protein annexin V binds preferentially to PS and ... More
Effects of permeation enhancers on the transport of a peptidomimetic thrombin inhibitor (CRC 220) in a human intestinal cell line (Caco-2).
AuthorsWerner U, Kissel T, Reers M
JournalPharm Res
PubMed ID8865316
PURPOSE: The effects of five different permeation enhancer systems on the transport properties of a peptidomimetic thrombin inhibitor. CRC 220, were investigated in monolayers of a human intestinal cell line (Caco-2). METHODS: The transepithelial transport rates and additionally the cytotoxic properties of these enhancers were characterized using the following tests: ... More
In vitro classical conditioning of abducens nerve discharge in turtles.
AuthorsKeifer J, Armstrong KE, Houk JC
JournalJ Neurosci
PubMed ID7623132
In vitro classical conditioning of abducens nerve activity was performed using an isolated turtle brainstem-cerebellum preparation by direct stimulation of the cranial nerves. Using a delayed training procedure, the in vitro preparation was presented with paired stimuli consisting of a 1 sec train stimulus applied to the auditory nerve (CS), ... More
Second harmonic and sum frequency generation imaging of fibrous astroglial filaments in ex vivo spinal tissues.
AuthorsFu Y, Wang H, Shi R, Cheng JX
JournalBiophys J
PubMed ID17293404
Sum frequency generation (SFG) and second harmonic generation (SHG) were observed from helical fibrils in spinal cord white matter isolated from guinea pigs. By combining SFG with coherent anti-Stokes Raman scattering microscopy, which allows visualization of myelinated axons, these fibers were found to be distributed near the surface of the ... More
The 43-kD polypeptide of heart gap junctions: immunolocalization, topology, and functional domains.
AuthorsYancey SB, John SA, Lal R, Austin BJ, Revel JP
JournalJ Cell Biol
PubMed ID2472402
Analysis by SDS-PAGE of gap junction fractions isolated from heart suggests that the junctions are comprised of a protein with an Mr 43,000. Antibodies against the electroeluted protein and a peptide representing the 20 amino terminal residues bind specifically on immunoblots to the 43-kD protein and to the major products ... More
Airway surface liquid osmolality measured using fluorophore-encapsulated liposomes.
AuthorsJayaraman S, Song Y, Verkman AS
JournalJ Gen Physiol
PubMed ID11331352
The airway surface liquid (ASL) is the thin layer of fluid coating the luminal surface of airway epithelial cells at an air interface. Its composition and osmolality are thought to be important in normal airway physiology and in airway diseases such as asthma and cystic fibrosis. The determinants of ASL ... More
Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis).
AuthorsDarzynkiewicz Z, Juan G, Li X, Gorczyca W, Murakami T, Traganos F
JournalCytometry
PubMed ID9000580
The term cell necrobiology is introduced to comprise the life processes associated with morphological, biochemical, and molecular changes which predispose, precede, and accompany cell death, as well as the consequences and tissue response to cell death. Two alternative modes of cell death can be distinguished, apoptosis and accidental cell death, ... More
Development of a streak-camera-based time-resolved microscope fluorimeter and its application to studies of membrane fusion in single cells.
AuthorsKusumi A, Tsuji A, Murata M, Sako Y, Yoshizawa AC, Kagiwada S, Hayakawa T, Ohnishi S
JournalBiochemistry
PubMed ID2054350
A time-resolved microscope fluorimeter based on a synchroscan streak camera and a fast pulsed laser system has been developed to measure the fluorescence lifetime decay under the fluorescence microscope. This system allows one to measure the nanosecond fluorescence lifetimes of fluorophores in a small spot (0.8-6.3 microns diameter) in single ... More
Antibody-bearing liposomes as multicolor immunofluorescence markers for flow cytometry and imaging.
AuthorsTruneh A, Machy P, Horan PK
JournalJ Immunol Methods
PubMed ID3110296
Liposomes covalently coupled to monoclonal antibodies retain the specificity of the antibody and bind only to cells bearing the appropriate determinant. As opposed to directly labeled antibodies which generally have fluorochrome to protein ratio of between 2-5, the entrapped space inside liposome can contain several hundred to several thousand molecules ... More
Detection of in situ activation of transglutaminase during apoptosis: correlation with the cell cycle phase by multiparameter flow and laser scanning cytometry.
AuthorsGrabarek J, Ardelt B, Kunicki J, Darzynkiewicz Z
JournalCytometry
PubMed ID12357464
BACKGROUND: One of the hallmarks of apoptosis is activation of tissue transglutaminase (Tgase; also called transglutaminase type 2 [TGase 2]). Its activation causes cross-linking of cytoplasmic proteins, making them insoluble and presumably less immunogenic. Several biochemical and cytochemical methods to detect activity of TGase 2 exist, but none has been ... More
Distribution and function of laminins in the neuromuscular system of developing, adult, and mutant mice.
AuthorsPatton BL, Miner JH, Chiu AY, Sanes JR
JournalJ Cell Biol
PubMed ID9396756
Laminins, heterotrimers of alpha, beta, and gamma chains, are prominent constituents of basal laminae (BLs) throughout the body. Previous studies have shown that laminins affect both myogenesis and synaptogenesis in skeletal muscle. Here we have studied the distribution of the 10 known laminin chains in muscle and peripheral nerve, and ... More
Deregulation of collagen phagocytosis in aging human fibroblasts: effects of integrin expression and cell cycle.
AuthorsLee W, McCulloch CA
JournalExp Cell Res
PubMed ID9434634
Intracellular degradation of collagen by phagocytosis in fibroblasts is essential for physiological remodeling of the extracellular matrix in a wide variety of connective tissues but imbalances between degradation and synthesis can lead to loss of tissue collagen. As aging is associated with loss of dermal and periodontal collagen and with ... More
Features of apoptotic cells measured by flow cytometry.
AuthorsDarzynkiewicz Z, Bruno S, Del Bino G, Gorczyca W, Hotz MA, Lassota P, Traganos F
JournalCytometry
PubMed ID1333943
The present review describes several methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. Most of these methods were applied to studies of apoptosis triggered in the human leukemic HL-60 cell line by DNA topoisomerase I or II inhibitors, and in rat thymocytes by ... More
Antimycin A-induced apoptosis of HL-60 cells.
AuthorsKing MA, Radicchi-Mastroianni MA
JournalCytometry
PubMed ID12442310
BACKGROUND: Previous experiments in our laboratory investigating apoptosis induced in HL-60 cells by camptothecin (CAM) have revealed that the sequence and rapidity of the apoptotic phenomena in an individual cell depend on the proliferative state of that cell when it encounters CAM. The role of mitochondria in HL-60 apoptosis was ... More
Comparison of cortically and subcortically controlled motor systems: I. Morphology of intracellularly filled rubrospinal neurons in rat and turtle.
AuthorsLustig DG, Herrick JL, Keifer J
JournalJ Comp Neurol
PubMed ID9651009
The rat and turtle differ markedly in major structural features of the corticocerebellorubrospinal circuitry. Although both species have a well-developed cerebellorubrospinal system, they differ in that a direct cerebral cortical input to the red nucleus is present only in the rat. The aim of the present study was to compare ... More
Calcium imaging in live rat optic nerve myelinated axons in vitro using confocal laser microscopy.
AuthorsRen Y, Ridsdale A, Coderre E, Stys PK
JournalJ Neurosci Methods
PubMed ID11040413
Intracellular Ca(2+) plays a major role in the physiological responses of excitable cells, and excessive accumulation of internal Ca(2+) is a key determinant of cell injury and death. Many studies have been carried out on the internal Ca(2+) dynamics in neurons. In constrast, there is virtually no such information for ... More
Pleated septate junctions in leech photoreceptors: ultrastructure, arrangement of septa, gate and fence functions.
AuthorsAschenbrenner S, Walz B
JournalCell Tissue Res
PubMed ID9662648
The leech photoreceptor forms a unicellular epithelium: every cell surrounds an extracellular "vacuole" that is connected to the remaining extracellular space via narrow clefts containing pleated septate junctions. We analyzed the complete structural layout of all septa within the junctional complex in elastic brightfield stereo electron micrographs of semithin serial ... More
Effects of caspase inhibition on camptothecin-induced apoptosis of HL-60 cells.
AuthorsKing MA, Radicchi-Mastroianni MA
JournalCytometry
PubMed ID12210608
BACKGROUND: During camptothecin (CAM)-induced apoptosis of HL-60 cells, the external exposure of phosphatidylserine (PS) can either precede or follow DNA cleavage. The evidence suggests that cells in S-phase when CAM is added undergo rapid DNA, nuclear, and cellular disintegration before exposing PS on the outside of the plasma membrane, whereas ... More
In vivo staining of oligodendroglia in the rabbit retina.
AuthorsEhinger B, Zucker CL, Bruun A, Adolph A
JournalGlia
PubMed ID7507886
We have discovered that a strongly fluorescent dye, sulforhodamine 101, when injected intravitreally in vivo, very effectively stains a class of star-shaped cells in the innermost layers of the rabbit retina. The cells were strictly confined to the region containing medullated fibers and emitted dichotomously branching processes that ended up ... More