CD5 antibodies increase intracellular ionized calcium concentration in T cells.
AuthorsJune CH,Rabinovitch PS,Ledbetter JA
JournalJournal of immunology (Baltimore, Md. : 1950)
PubMed ID3106489
The binding of a variety of monoclonal antibodies to the CD5 (T, gp67) pan T cell differentiation antigen has been shown to potentiate T cell proliferation. In this paper we show that CD5 monoclonal antibodies cause increased intracellular free calcium concentration ([Ca2+]i) in T cells. An increase in [Ca2+]i occurred ... More
Transcriptional activation of HLA-DR alpha by interferon gamma requires a trans-acting protein.
AuthorsBlanar MA, Boettger EC, Flavell RA
JournalProc Natl Acad Sci U S A
PubMed ID3133656
Stimulation of the human epithelial-like cell line, HeLa, with interferon gamma (IFN-gamma) induces steady-state levels of HLA-DR alpha mRNA. Using a sensitive RNase-mapping procedure, we detect induced HLA-DR alpha mRNA as early as 8 hr after IFN-gamma treatment; maximal accumulation occurs by 48 hr. Treatment with the protein synthesis inhibitor, ... More
TCR engagement of CD4+CD8+ thymocytes in vitro induces early aspects of positive selection, but not apoptosis.
AuthorsGroves T, Parsons M, Miyamoto NG, Guidos CJ
JournalJ Immunol
PubMed ID8977176
'Immature CD4/CD8 double-positive (DP) thymocytes expressing self MHC-restricted TCR are positively selected in response to TCR signals to survive and differentiate into functionally competent CD4 or CD8 single positive (SP) T cells. In contrast, DP precursors expressing autoreactive TCR are clonally deleted in response to TCR signals. We show here ... More
ATP-dependent association of nuclear proteins with isolated rat liver nuclei.
AuthorsImamoto-Sonobe N, Yoneda Y, Iwamoto R, Sugawa H, Uchida T
JournalProc Natl Acad Sci U S A
PubMed ID3368451
'In vitro association of Xenopus nucleoplasmin and mammalian nonhistone chromosomal high mobility group 1 (HMG1) protein with nuclei isolated from rat liver was examined. Efficient association of nuclear proteins with isolated nuclei requires ATP, HCO3-, and Ca2+. Association occurred at 33 degrees C but not at 4 degrees C. ATP ... More
Antioxidant activity of metallothionein compared with reduced glutathione.
AuthorsMiura T, Muraoka S, Ogiso T
JournalLife Sci
PubMed ID9155005
'The antioxidant activity of metallothionein (MT) was investigated. Metallothionein scavenged hydroxyl radicals (HO.) to protect DNA from the oxidative attack by microsomes. This scavenging activity of MT was approximately 50 times greater than reduced glutathione (GSH) on a molar basis. However, MT did not inhibit DNA damage induced by microsomes ... More
Distinct patterns of transmembrane calcium flux and intracellular calcium mobilization after differentiation antigen cluster 2 (E rosette receptor) or 3 (T3) stimulation of human lymphocytes.
AuthorsJune CH, Ledbetter JA, Rabinovitch PS, Martin PJ, Beatty PG, Hansen JA
JournalJ Clin Invest
PubMed ID2420827
'We evaluated CD2 (E rosette) and CD3 (T3)-triggered activation of resting lymphocytes by measuring the intracellular free calcium concentration ([Ca2+]i) of individual cells. The [Ca2+]i of indo-1-loaded cells was measured by flow cytometry and responses were correlated with cell surface phenotype. Stimulation with anti-CD3 antibody caused an increase in [Ca2+]i ... More
Two-color flow cytometric analysis of monocyte depleted human blood lymphocyte subsets.
AuthorsFleisher TA, Marti GE, Hagengruber C
JournalCytometry
PubMed ID3261232
'Peripheral blood mononuclear cells (PBMC) from normal individuals were examined using 16 pairs of FITC and phycoerythrin (PE) directly conjugated monoclonal antibodies. Each pair of reagents was used to evaluate a conventional lymphocyte gate as well as open (non) gate of monocyte depleted PBMC. Parallel studies using the same panel ... More
Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements.
AuthorsPrior RL, Wu X, Schaich K,
JournalJ Agric Food Chem
PubMed ID15884874
'Methods available for the measurement of antioxidant capacity are reviewed, presenting the general chemistry underlying the assays, the types of molecules detected, and the most important advantages and shortcomings of each method. This overview provides a basis and rationale for developing standardized antioxidant capacity methods for the food, nutraceutical, and ... More
Novel fluorescein-based flow-cytometric method for detection of lipid peroxidation.
AuthorsMakrigiorgos GM, Kassis AI, Mahmood A, Bump EA, Savvides P
JournalFree Radic Biol Med
PubMed ID8958133
'The novel property of fluorescein to detect peroxyl radicals is demonstrated. On the basis of this observation, a fluorescein-based, flow-cytometric method to directly and continuously detect free radicals generated in cell membranes during lipid peroxidation has been developed. 5- and 6-Carboxyfluorescein (5-/6-CF) free in solution and fluorescein-labeled polylysine lose their ... More
The mechanism of nuclear transport of natural or artificial transport substrates in digitonin-permeabilized cells.
AuthorsCserpán I, Udvardy A
JournalJ Cell Sci
PubMed ID7657709
'Characterization of nuclear protein transport in digitonin-permeabilized cells revealed that the number of the nuclear localization signal sequences (NLS) within the transport substrate basically influences the mechanism of the transport reaction. Phycoerythrine-NLS transport substrate carrying a maximum of 4-5 conjugated NLSs/subunit, or Bsp methyltransferase-NLS fusion protein were efficiently transported into ... More
Mobility of cell surface receptors: a re-evaluation.
AuthorsCherry RJ, Smith PR, Morrison IE, Fernandez N
JournalFEBS Lett
PubMed ID9678600
'It has long been known from fluorescence recovery after photobleaching experiments that the mobility of most cell surface receptors is much smaller than expected for free diffusion of proteins in a fluid lipid bilayer. Single-particle tracking experiments are currently revealing the complexity of the constraints to free diffusion. Evidence has ... More
HEMCAM, an adhesion molecule expressed by c-kit+ hemopoietic progenitors.
AuthorsVainio O, Dunon D, Aïssi F, Dangy JP, McNagny KM, Imhof BA
JournalJ Cell Biol
PubMed ID8978830
'We have characterized the adhesion molecule HEMCAM, which is expressed by hemopoietic progenitors of embryonic bone marrow. HEMCAM belongs to the immunoglobulin superfamily and consists of the V-V-C2-C2-C2 Ig domains. There are three mRNA splice variants. One has a short cytoplasmic tail; another has a long tail; while the third ... More
Use of phycoerythrin and allophycocyanin for fluorescence resonance energy transfer analyzed by flow cytometry: advantages and limitations.
AuthorsBatard P, Szollosi J, Luescher I, Cerottini J-C, MacDonald R, Romero P
JournalCytometry
PubMed ID12116371
'This study validates the use of phycoerythrin (PE) and allophycocyanin (APC) for fluorescence energy transfer (FRET) analyzed by flow cytometry.'
Oxidant stress in nephrotic syndrome: comparison of F(2)-isoprostanes and plasma antioxidant potential.
AuthorsDogra G, Ward N, Croft KD, Mori TA, Barrett PH, Herrmann SE, Irish AB, Watts GF
JournalNephrol Dial Transplant
PubMed ID11477165
'BACKGROUND: The nephrotic syndrome (NS) is associated with an increased risk of coronary heart disease. Increased oxidant stress may contribute to this by means of hyperlipidaemia and/or hypoalbuminaemia. In this study we assessed the contributory role of oxidant stress, as measured by F(2)-isoprostanes and plasma oxygen radical absorbance capacity (ORAC), ... More
Time-resolved detection of energy transfer: theory and application to immunoassays.
AuthorsMorrison LE
JournalAnal Biochem
PubMed ID3218725
'Energy-transfer measurements based upon acceptor fluorophore emission are plagued with background fluorescence resulting from absorption of the excitation light by the acceptor fluorophore. The present work examines the use of a long-lifetime donor fluorophore and a short-lifetime acceptor fluorophore, combined with pulsed-laser excitation and electronic gating of detector signals, to ... More
Human lymphocytes making rheumatoid factor and antibody to ssDNA belong to Leu-1+ B-cell subset.
AuthorsCasali P, Burastero SE, Nakamura M, Inghirami G, Notkins AL
JournalScience
PubMed ID3105056
'B lymphocytes bearing the Leu-1 cell-surface antigen (Leu-1+), the human equivalent of mouse Ly-1+ B lymphocytes, have been detected in human peripheral blood, but there is little information on their frequency and properties. Analysis by fluorescence-activated cell sorter and double immunofluorescence showed that Leu-1+ B cells are consistently present in ... More
Cytofluorometric analyses of human T cell CD2/CD4 inter-molecular interactions.
AuthorsBueso-Ramos CE, Donahoe RM, Nicholson JK, Madden JJ, Falek A
JournalJ Immunol
PubMed ID2964474
'Incubation of human T lymphocytes with saturating concentrations of combinations of certain anti-CD2 and -CD4 mAb results in reciprocal down-regulation of the cell surface density expression of the respective CD molecules. Such reciprocal down-regulation occurs at 0 degrees C in the presence of sodium azide and appears selective for CD2 ... More
Resolving leukocytes using axial light loss.
AuthorsStewart CC, Stewart SJ, Habbersett RC
JournalCytometry
PubMed ID2766889
'Axial light loss (ALL) is the measurement of the total light lost from the laser beam at 0 degrees when a particle passes through the beam. Used in combination with the monoclonal antibody CD45, ALL can effectively resolve lymphocytes, monocytes, granulocytes, and dead cells in viable or fixed preparations of ... More
Phycoerythrin fluorescence-based assay for peroxy radicals: a screen for biologically relevant protective agents.
AuthorsDeLange RJ, Glazer AN
JournalAnal Biochem
PubMed ID2567129
'Under the conditions of this assay, antioxidants that react rapidly with peroxy free radicals (e.g., ascorbate, vitamin E analogs, urate), protect phycoerythrin completely from damage by such radicals generated by thermal decomposition of 2,2''-azobis(2-amidinopropane); other compounds provide partial concentration-dependent protection. Change in phycoerythrin fluorescence emission with time provides a measure ... More
Lipid peroxidation induced by phenylbutazone radicals.
AuthorsMiura T, Muraoka S, Fujimoto Y
JournalLife Sci
PubMed ID12269388
'Lipid peroxidation was investigated to evaluate the deleterious effect on tissues by phenylbutazone (PB). PB induced lipid peroxidation of microsomes in the presence of horseradish peroxidase and hydrogen peroxide (HRP-H2O2). The lipid peroxidation was completely inhibited by catalase but not by superoxide dismutase. Mannitol and dimethylsulfoxide had no effect. These ... More
The use of fluorescent probes in immunochemistry.
AuthorsDavidson RS, Hilchenbach MM
JournalPhotochem Photobiol
PubMed ID2217553
'The limitations and advantages of particular dyes for labelling proteins and other biological materials are discussed. Methods available for conjugating dyes to proteins are outlined. Following a discussion of double labelling methods the use of photoactivatable fluorochromes and time resolved fluorescence methodologies are outlined. The reasons for the photoinstability of ... More
'Nuclear pore proteins bearing O-linked N-acetylglucosamine (GlcNAc) are involved in nuclear transport, although a role for their glycosylation is not established. Xenopus egg extracts capable of reforming functional nuclei in vitro yielded nuclei with impaired transport and reduced nuclear pore density when depleted of wheat germ agglutinin-binding proteins. Many of ... More
Automated assay of oxygen radical absorbance capacity with the COBAS FARA II.
AuthorsCao G, Verdon CP, Wu AH, Wang H, Prior RL
JournalClin Chem
PubMed ID7497614
'Reactive oxygen species are of interest in biology and medicine because of evidence relating them to aging and disease processes. A relatively simple but sensitive and reliable method for quantifying the oxygen radical absorbance capacity (ORAC) of antioxidants in biological tissues has been automated for use with the COBAS FARA ... More
Phycobiliproteins.
AuthorsGlazer AN
JournalMethods Enzymol
PubMed ID3148835
Coupling of monoclonal antibodies with fluorophores.
AuthorsHaugland RP
JournalMethods Mol Biol
PubMed ID7550684
Fluorescence-activated cell sorting: theory, experimental optimization, and applications in lymphoid cell biology.
AuthorsParks DR, Herzenberg LA
JournalMethods Enzymol
PubMed ID6396481
Comparative biochemistry of photosynthetic light-harvesting systems.
AuthorsGlazer AN
JournalAnnu Rev Biochem
PubMed ID6412621
Light harvesting by phycobilisomes.
AuthorsGlazer AN
JournalAnnu Rev Biophys Biophys Chem
PubMed ID3924069
The use of phycobiliproteins as fluorescent labels in immunoassay.
AuthorsKronick MN
JournalJ Immunol Methods
PubMed ID3528294
Light guides. Directional energy transfer in a photosynthetic antenna.
AuthorsGlazer AN
JournalJ Biol Chem
PubMed ID2491842
Protein labeling with fluorescent probes.
AuthorsHolmes KL, Lantz LM
JournalMethods Cell Biol
PubMed ID11060842
Phycoerythrin fluorescence-based assay for reactive oxygen species.
AuthorsGlazer AN,
JournalMethods Enzymol
PubMed ID2233292
The fluorescence of phycobiliproteins is highly sensitive to the conformation and chemical integrity of the protein and of the prosthetic groups. Under appropriate conditions, in the presence of reactive oxygen species, the rate of loss of phycoerythrin fluorescence is an index of free radical damage. The effect of added compounds ... More
Multiparameter analysis of leukocytes by flow cytometry.
AuthorsStewart CC, Stewart SJ
JournalMethods Cell Biol
PubMed ID7861980
The flow cytometry described can be performed using a single laser. Each laboratory has to establish its own experience base and standard operating procedures. The intent of this discussion has been to illustrate the procedures that will lead to good flow cytometry data acquisition and analysis and to illustrate problematic ... More
Indirect immunofluorescence labelling with complexes of phycoerythrin and monoclonal anti-phycoerythrin antibodies (PEAPE complexes).
AuthorsGuy K, Crichton DN, Ross JA
JournalJ Immunol Methods
PubMed ID3047264
We describe a method of immunofluorescence which is a lateral application of the principles of the APAAP immunohistochemical technique. Immune complexes of R-phycoerythrin and monoclonal anti-R-phycoerythrin (PEAPE complexes) were used in an indirect immunofluorescence technique to detect the binding to cells of monoclonal antibodies directed to IgM, HLA-DR and B ... More
Immunoassay techniques with fluorescent phycobiliprotein conjugates.
AuthorsKronick MN, Grossman PD
JournalClin Chem
PubMed ID6883673
We describe immunoassay techniques that take advantage of the novel properties of phycobiliprotein fluorescent dyes. These dyes, which can be isolated from a wide variety of algae, exhibit extremely high absorptivities, high quantum efficiencies, and excitation and emission bands across the visible spectrum. These stable, hydrophilic proteins can easily be ... More
Separation of phycobiliprotein subunits by reverse-phase high-pressure liquid chromatography.
AuthorsSwanson RV, Glazer AN
JournalAnal Biochem
PubMed ID2221378
Baseline separation of subunits of diverse phycobiliproteins was achieved by a reverse-phase HPLC gradient method with a C4 large-pore column and a solvent system consisting of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in 2:1 (v/v) acetonitrile:isopropanol. The procedure was successfully applied to cyanobacterial allophycocyanin and C-phycocyanins, an ... More
Rapid detection of low levels of donor specific IgG by flow cytometry with single and dual colour fluorescence in renal transplantation.
AuthorsTalbot D, Givan AL, Shenton BK, Stratton A, Proud G, Taylor RM
JournalJ Immunol Methods
PubMed ID3047265
Lymphocytotoxic immunoglobulin is routinely assayed before human renal transplantation. If IgG directed against donor T cells is detected in the serum of the potential recipient, transplantation is not performed as it is associated with a poor graft outcome. Poor sensitivity of the conventional assay has been postulated as being the ... More
Photostability studies of phycobiliprotein fluorescent labels.
AuthorsWhite JC, Stryer L
JournalAnal Biochem
PubMed ID3578804
Photostability studies of four fluorescent phycobiliproteins were conducted to identify stable chromophores for biological labeling applications. Phycobiliprotein photodestruction was linear with the applied laser power and depended on the total number of photons absorbed per molecule. Photodestruction quantum yields phi of 1.1 X 10(-5) for R-phycoerythrin, 6.6 X 10(-6) for ... More
Efficient use of monoclonal antibodies for immunofluorescence.
AuthorsEdwards BS, Shopp GM
JournalCytometry
PubMed ID2465115
We describe here a simple and rapid small volume microplate-based immunofluorescence staining method in which fluorochrome-conjugated monoclonal antibodies (MAb) from three different manufacturers, used at a single standardized quantity (50 ng per test), resulted in optimal staining of human lymphocyte subsets. Staining reactions were robust, in that the number of ... More
We identified intracellular adhesion molecule-2 (ICAM-2) in a genetic screen as an activator of the PI3K/AKT pathway leading to inhibition of apoptosis. ICAM-2 induced tyrosine phosphorylation of ezrin and PI3K kinase membrane translocation, resulting in phosphatidylinositol 3,4,5 production, PDK-1 and AKT activation, and subsequent phosphorylation of AKT targets BAD, GSK3, ... More
Electropore diameters, lifetimes, numbers, and locations in individual erythrocyte ghosts.
AuthorsSowers AE, Lieber MR
JournalFEBS Lett
PubMed ID3743774
Low light level video microscopy was used to study the diameter, lifetime, number, and location characteristics of electric field-induced pores (electropores) in erythrocyte ghosts. The diameter of electropores was probed by following the efflux of soluble fluorescent-tagged molecules out of the resealed ghost cytoplasmic compartments. After reaching a peak radius ... More
Simultaneous identification of eight leucocyte subsets of human peripheral blood using three-colour immunofluorescence flow cytometric analysis.
AuthorsCarayon P, Bord A, Raymond M
JournalJ Immunol Methods
PubMed ID2033277
The full analytical potential of flow cytometry has been exploited in order to identify a maximum of human peripheral blood leucocyte subsets in a single tube. For this purpose a mixture composed of six different monoclonal antibodies directly coupled to FITC or phycoerythrin or indirectly linked to the DuoChrome reagent ... More
Discrimination of viable and non-viable cells using propidium iodide in two color immunofluorescence.
AuthorsSasaki DT, Dumas SE, Engleman EG
JournalCytometry
PubMed ID3113897
The relative ease with which a flow cytometer can perform simultaneous two color immunofluorescence to examine subpopulations of lymphoid cells has been well documented. Thus, flow cytometers equipped with only a single argon laser can be used to delineate various cell types by exciting both fluorescein- and phycoerythrin-conjugated antibodies to ... More
Ultrasensitive native fluorescence detection of proteins with miniaturized polyacrylamide gel electrophoresis by laser side-entry excitation.
AuthorsZhang H, Yeung ES
JournalElectrophoresis
PubMed ID16927346
Direct detection of separated proteins inside polyacrylamide gels has many advantages compared to staining methods. Ultrasensitive native fluorescence detection of proteins with miniaturized 1-D and 2-D PAGE was achieved with laser side-entry excitation. The detection limit for R-phycoerythrin protein spots in 1-D SDS-PAGE with 532 nm excitation was as low ... More
Fixation and long-term storage of human lymphocytes for surface marker analysis by flow cytometry.
AuthorsLal RB, Edison LJ, Chused TM
JournalCytometry
PubMed ID3132354
A method to preserve stained human lymphocytes for subsequent cell surface analysis by flow cytometry (FCM) is described. Cells stained with fluorescein isothiocyanate (FITC) and phycoerythrin (PE)-conjugated monoclonal antibodies and then fixed in 1% paraformaldehyde, followed by extensive washing and resuspension in 1% BSA medium, could be stored at 4 ... More
Fluorescence-based assay for reactive oxygen species: a protective role for creatinine.
AuthorsGlazer AN
JournalFASEB J
PubMed ID3371593
Attack by reactive oxygen species leads to a decay in phycoerythrin fluorescence emission. This phenomenon provides a versatile new assay for small molecules and macromolecules that can function as protective compounds. With 1-2 x 10(-8) M phycoerythrin, under conditions where peroxyl radical generation is rate-limiting, the fluorescence decay follows apparent ... More
Endocytosis of beta 2 integrins by stimulated human neutrophils analyzed by flow cytometry.
AuthorsChambers JD, Simon SI, Berger EM, Sklar LA, Arfors KE
JournalJ Leukoc Biol
PubMed ID8097762
Flow cytometry and fluorescently labeled monoclonal antibodies were used to investigate endocytosis of human neutrophil beta 2 integrins following cellular activation. CD18 initially present on the cell surface cycled in two phases after exposure to formyl peptide or platelet-activating factor. The first phase lasted 3 min at 37 degrees C; ... More
Five-dimensional flow cytometry as a new approach for blood and bone marrow differentials.
AuthorsTerstappen LW, Loken MR
JournalCytometry
PubMed ID3208621
We have used five independent variables on a flow cytometer to discriminate and to quantify the cellular components within both blood and bone marrow aspirates. The signals were stored in list mode by which a five-dimensional space was created. The cells--differentiated into: 1) erythrocytes, 2) reticulocytes, 3) nucleated erythroid cells, ... More
Dynamics of single-protein molecules at a liquid/solid interface: implications in capillary electrophoresis and chromatography.
AuthorsKang SH, Yeung ES
JournalAnal Chem
PubMed ID12510756
The behavior of individual molecules of R-phycoerythrin (RPE) was monitored by fluorescence imaging at various pHs and ionic strengths within the evanescent-field layer (EFL) at a water/fused-silica interface. Above the isoelectric point (pI), the individual protein molecules moved between exposures with random motion. As the pH approached the pI of ... More
Antigen-independent regulation of cytoplasmic calcium in B cells with a 12-kDa B-cell growth factor and anti-CD19.
AuthorsLedbetter JA, Rabinovitch PS, June CH, Song CW, Clark EA, Uckun FM
JournalProc Natl Acad Sci U S A
PubMed ID2450350
Increases in cytoplasmic free calcium ([Ca2+]i) can be induced in resting B cells either by a low molecular weight (12-kDa) B-cell growth factor (LMW-BCGF) or by crosslinking the B-cell antigen CD19 with monoclonal antibody (mAb). LMW-BCGF causes a slow [Ca2+]i increase in peripheral blood and tonsillar B cells but has ... More
Rapid screening and selection of monoclonal antibodies by bivariate flow cytometric analyses.
AuthorsPallavicini MG, Haendel S, Rosette C, Barlett R
JournalJ Immunol Methods
PubMed ID2464042
We describe a bivariate flow cytometric assay to rapidly identify hybridomas producing new monoclonal antibodies recognizing subpopulations that are unreactive with existing immunological reagents. In this screen, whole cells in microtiter wells are labeled first with a red-linked test antibody, and then with a green-linked cocktail of existing monoclonal antibody ... More
AuthorsSiiman O, Wilkinson J, Burshteyn A, Roth P, Ledis S
JournalBioconjug Chem
PubMed ID10563780
New, highly amino-substituted dextran or aminodextran (hereafter denoted Amdex) of various sizes between about 20 and 1000 kDa molecular mass and degrees of amino-substitution between 7 and 40% were prepared and characterized by elemental analyses and polyacrylamide gel electrophoresis. These aminodextrans together with others commercially available were shown by static ... More
A modular detector for flow cytometric multicolor fluorescence measurements.
AuthorsSteinkamp JA, Habbersett RC, Stewart CC
JournalCytometry
PubMed ID3622158
A modular detector for measuring multicolor fluorescence from cells illuminated by single or multiple lasers has been developed for flow cytometers. Motion picture projector, camera, and CCTV/video lenses were evaluated for use in the detector by comparing their physical characteristics, image quality, and light collection efficiencies. A 25-mm focal length ... More
Tracking single proteins within cells.
AuthorsGoulian M, Simon SM
JournalBiophys J
PubMed ID11023923
We present experiments in which single proteins were imaged and tracked within mammalian cells. Single proteins of R-phycoerythrin (RPE) were imaged by epifluorescence microscopy in the nucleoplasm and cytoplasm at 71 frames/s. We acquired two-dimensional trajectories of proteins (corresponding to the projection of three-dimensional trajectories onto the plane of focus) ... More
Novel roles of CpG oligodeoxynucleotides as a leader for the sampling and presentation of CpG-tagged antigen by dendritic cells.
AuthorsShirota H, Sano K, Hirasawa N, Terui T, Ohuchi K, Hattori T, Shirato K, Tamura G
JournalJ Immunol
PubMed ID11418633
Oligodeoxynucleotides containing CpG motifs have been highlighted as potent Th1 activators. We previously reported that Ag and CpG, when conjugated together, synergistically promoted the Ag-specific Th1 development and inhibited the Th2-mediated airway eosinophilia. In this study, we examined the mechanisms underlying the synergism of the covalent conjugation. The CpG-OVA conjugate ... More
Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with phycoerythrin.
AuthorsPeck K, Stryer L, Glazer AN, Mathies RA
JournalProc Natl Acad Sci U S A
PubMed ID2726766
A theory for single-molecule fluorescence detection is developed and then used to analyze data from subpicomolar solutions of B-phycoerythrin (PE). The distribution of detected counts is the convolution of a Poissonian continuous background with bursts arising from the passage of individual fluorophores through the focused laser beam. The autocorrelation function ... More
Immunofluorescent labeling using covalently linked anti-phycoerythrin antibodies and phycoerythrin polymers.
AuthorsWilson MR, Crowley S, Odgers GA, Shaw L
JournalCytometry
PubMed ID2065561
We have developed methods for covalently attaching anti-phycoerythrin (PE) MAbs to other antibodies, and for using PE or polymers of PE in conjunction with these conjugates to rapidly produce specific, high intensity fluorescent labeling of antigens. The performance of these systems was examined on the surface of microspheres and on ... More
A practical approach to multicolor flow cytometry for immunophenotyping.
AuthorsBaumgarth N, Roederer M
JournalJ Immunol Methods
PubMed ID10986408
Through a series of novel developments in flow cytometry hardware, software, and dye-chemistry it is now possible to simultaneously measure up to 11 distinct fluorescences and two scattered light parameters on each cell. Such advanced multicolor systems have a number of advantages over current two- and three-color flow cytometric measurements. ... More
8 color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity.
AuthorsRoederer M, De Rosa S, Gerstein R, Anderson M, Bigos M, Stovel R, Nozaki T, Parks D, Herzenberg L, Herzenberg L
JournalCytometry
PubMed ID9415416
We developed the chemistry, instrumentation, and software technologies needed to measure, simultaneously and independently, eight different fluorescent molecules on individual cells. Conjugation of these fluorochromes to monoclonal antibodies is straightforward; all immunofluorescence staining is accomplished with direct stains only. We built a hybrid flow cytometer with eight fluorescence detectors and ... More
Simultaneous measurement of multiple active kinase states using polychromatic flow cytometry.
AuthorsPerez OD, Nolan GP
JournalNat Biotechnol
PubMed ID11821861
Intracellular assays of signaling systems have been limited by an inability to correlate functional subsets of cells in complex populations on the basis of active kinase states. Such correlations could be important in distinguishing changes in signaling status that arise in rare cell subsets during functional activation or in disease ... More
Visualization and tracking of single protein molecules in the cell nucleus.
AuthorsKues T, Peters R, Kubitscheck U
JournalBiophys J
PubMed ID11371468
A recently developed laser fluorescence videomicroscopy method was used to determine for the first time the intranuclear trajectories of single protein molecules. Using the recombinant Escherichia coli beta-galactosidase protein P4K, labeled with an average of 4.6 ALEXA 488 chromophores per tetramer, single P4K molecules could be localized and tracked in ... More
Concomitant delineation of surface Ig, B-cell differentiation antigens, and HLADR on lymphoid proliferations using three-color immunocytometry.
AuthorsSegal GH, Edinger MG, Owen M, McNealis M, Lopez P, Perkins A, Linden MD, Fishleder AJ, Stoler MH, Tubbs RR
JournalCytometry
PubMed ID1712263
Accurate and consistent enumeration of B-cell subpopulations in lymphoid tissue was achieved through multiparameter three-color immunofluorescence and flow cytometric analysis (FCM). Phycoerythrin (PE)-anti-CD19 (Leu12) and biotinylated anti-HLADr/streptavidin-Duochrome (PE/Texas Red), used in conjunction with polyclonal fluorescein isothiocyanate (FITC) conjugated anti-surface immunoglobulin (SIg) antibodies, effectively separated non-specific binding and background fluorescence from ... More
The gamma subunit of R-phycoerythrin and its possible mode of transport into the plastid of red algae.
AuthorsApt KE, Hoffman NE, Grossman AR
JournalJ Biol Chem
PubMed ID8344905
R-phycoerythrin is the major light-harvesting pigment protein of most red algal phycobilisomes. It is composed of three pigmented polypeptide subunits, the alpha, beta, and gamma. While alpha and beta phycoerythrin subunits are each unique in the red alga Aglaothamnion neglectum, there are two different gamma subunits with distinct molecular masses. ... More
Two- and three-color immunofluorescence using aminocoumarin, fluorescein, and phycoerythrin-labelled antibodies and single laser flow cytometry.
AuthorsDelia D, Martinez E, Fontanella E, Aiello A
JournalCytometry
PubMed ID1764977
Antibodies coupled to 7-aminocoumarin (AMCA) emit a bright blue fluorescence under ultraviolet (UV) excitation and are therefore ideal for three-color immunofluorescence (IF) with fluorescein (FITC) and phycoerythrin (PE) labeled reagents; however, due to the different absorption spectra, the use of these fluorophores for multicolor flow-cytometric analysis requires a double light ... More
A multistate model for the fluorescence response of R-phycoerythrin.
AuthorsGaigalas A, Gallagher T, Cole KD, Singh T, Wang L, Zhang YZ
JournalPhotochem Photobiol
PubMed ID16420100
Although strong fluorescence makes the R-phycoerythrin (R-PE) proteins increasingly useful in biological and clinical assays, they are subject to nonlinear effects including transitions to collective dark states and photodegradation, which complicate quantitative applications. We report measurements of R-PE fluorescence intensity as a function of incident power, duration of illumination and ... More
A fluorescence-based method for measuring total plasma antioxidant capability.
AuthorsGhiselli A, Serafini M, Maiani G, Azzini E, Ferro-Luzzi A
JournalFree Radic Biol Med
PubMed ID7896168
The Total Radical-Trapping Antioxidant Parameter (TRAP) of 10 freshly prepared human plasmas was measured by a new fluorometric assay. In this method, the rate of peroxidation induced by 2,2'-diazobis (2-amidinopropane) dihydrochloride (ABAP) was monitored through the loss of fluorescence of the protein R-Phycoerythrin (R-PE). The lag-phase induced by plasma was ... More
Leu 23 induction as an early marker of functional CD3/T cell antigen receptor triggering. Requirement for receptor cross-linking, prolonged elevation of intracellular [Ca++] and stimulation of protein kinase C.
AuthorsTesti R, Phillips JH, Lanier LL
JournalJ Immunol
PubMed ID2466079
The Leu 23 Ag is a phosphorylated 28 to 32-kDa disulfide-linked homodimer expressed on the surface of activated T cells, B cells, and NK cells. Resting, unstimulated peripheral blood T cells are Leu 23-, but 20 to 30% of normal thymocytes constitutively express Leu 23. Triggering of the CD3/TCR complex ... More
Functional competency of T cell antigen receptors in human thymus.
AuthorsWeiss A, Dazin PF, Shields R, Fu SM, Lanier LL
JournalJ Immunol
PubMed ID3500209
The T cell antigen receptor is likely to play a role in both positive and negative selection in the thymus. Three populations of thymocytes can be distinguished by the level of expression of the CD3-alpha/beta-chain heterodimer of the T cell antigen receptor (CD3/Ti alpha/beta) complex. Cells which fail to express ... More
Enhanced transmembrane signalling activity of monoclonal antibody heteroconjugates suggests molecular interactions between receptors on the T cell surface.
AuthorsLedbetter JA, Norris NA, Grossmann A, Grosmaire LS, June CH, Uckun FM, Cosand WL, Rabinovitch PS
JournalMol Immunol
PubMed ID2918858
Signal transduction occurs through multiple receptors expressed on mature, resting T cells. In addition to the CD3-T cell receptor complex, the CD2, CD4, CD5, CD7, CD8 and CD28 receptors mobilize cytoplasmic calcium within minutes of binding with monoclonal antibodies and additional crosslinking occurs on the cell surface. As an approach ... More
Single laser flow cytometric detection of lymphocytes binding three antibodies labelled with fluorescein, phycoerythrin and a novel tandem fluorochrome conjugate.
AuthorsFestin R, Björkland A, Tötterman TH
JournalJ Immunol Methods
PubMed ID2137494
In order to implement three-color surface immunofluorescence on a single laser flow cytometer, we combined a new fluorescent tandem conjugate (TC, R-phycoerythrin plus Texas red, available as DuoCHROME) with fluorescein (FITC)- and phycoerythrin (PE)-labelled monoclonal antibodies for the simultaneous detection of three antigens on individual lymphoid cells. Considerable amounts of ... More
Fluorescent phycobiliprotein conjugates for analyses of cells and molecules.
AuthorsOi VT, Glazer AN, Stryer L
JournalJ Cell Biol
PubMed ID6749865
The synthesis of a novel class of reagents for fluorescence analyses of molecules and cells is reported. These compounds consist of a highly fluorescent phycobiliprotein conjugated to a molecule having biological specificity. Phycoerythrin-immunoglobulin, phycoerythrin-protein A, and phycoerythrin-avidin conjugates were prepared. These conjugates bind specifically to beads containing a covalently attached ... More
Detection of triple antibody-binding lymphocytes in standard single laser flow cytometry using colloidal gold, fluorescein and phycoerythrin as labels.
AuthorsFestin R, Björklund B, Tötterman TH
JournalJ Immunol Methods
PubMed ID2956330
Goat anti-mouse antibodies conjugated with colloidal 40 nm gold particles (G40) were used as secondary layers to stain human T lymphocytes in an attempt to extend the polarized light epi-illumination microscopic technique to flow cytometric multiparameter analysis. G40-labelled T cells were further stained with phycoerythrin (PE, red)- and fluorescein (FITC, ... More
Phycoerythrin-allophycocyanin: a resonance energy transfer fluorochrome for immunofluorescence.
AuthorsTjioe I, Legerton T, Wegstein J, Herzenberg LA, Roederer M
JournalCytometry
PubMed ID11309805
BACKGROUND: As immunofluorescence experiments become more complex, the demand for new dyes with different properties increases. Fluorescent dyes with large Stoke's shifts that are very bright and have low background binding to cells are especially desirable. We report on the properties of the resonance energy tandems of phycoerythrin and allophycocyanin ... More
Nucleocytoplasmic protein traffic in single mammalian cells studied by fluorescence microphotolysis.
AuthorsSchulz B, Peters R
JournalBiochim Biophys Acta
PubMed ID3651509
Fluorescence microphotolysis was employed to measure in single living cells the kinetics of nucleocytoplasmic transport and the coefficients of intracellular diffusional mobility for the nuclear non-chromosomal protein nucleoplasmin. Nucleoplasmin was isolated from Xenopus ovary and labeled fluorescently. By injection into Xenopus oocytes it was ascertained that fluorescent labeling did not ... More
Lymphocyte surface thiol levels.
AuthorsSahaf B, Heydari K, Herzenberg LA, Herzenberg LA
JournalProc Natl Acad Sci U S A
PubMed ID12642656
Recent studies have implicated reduced thiols (cysteine -SH) in the function of individual cell surface proteins. Studies presented here demonstrate that the overall level of reduced thiols on cell surface molecules differs on individual subsets of peripheral blood mononuclear cells and that these levels can be manipulated in vitro by ... More
Cyclosporin A does not inhibit the PHA-stimulated increase in intracellular Ca2+ concentration but inhibits the increase in E-rosette receptor (CD2) expression and appearance of interleukin-2 receptors (CD25).
AuthorsRedelman D
JournalCytometry
PubMed ID2896111
The immunosuppressive drug cyclosporin A (CsA) inhibits mixed lymphocyte responses, blocks the generation of cytotoxic T lymphocytes, and inhibits the T lymphocyte proliferative response stimulated by polyclonal activators such as phytohemagglutinin (PHA). Nevertheless, there have been contradictory reports attempting to explain the mechanism(s) for this immunosuppressive activity. In the current ... More
Simultaneous measurement of two cellular antigens and DNA using fluorescein-isothiocyanate, R-phycoerythrin, and propidium iodide on a standard FACScan.
AuthorsCorver WE, Cornelisse CJ, Fleuren GJ
JournalCytometry
PubMed ID8168399
Multiparameter flow cytometry is a powerful tool for analyzing the phenotypic, cell kinetic, and ploidy heterogeneity of tumor cell populations. Because of the substantial spectral overlap of propidium iodide (PI) and R-phycoerythrin (PE) fluorescence emission, this combined use of these fluorochromes has been thought not to be feasible on a ... More
A long-lived fusogenic state is induced in erythrocyte ghosts by electric pulses.
AuthorsSowers AE
JournalJ Cell Biol
PubMed ID3958050
Treatment of erythrocyte ghosts in random positions in a suspension with membrane fusion-inducing direct current electric field pulses causes the membranes to become fusogenic. Significant fusion yields are observed if the membranes are dielectrophoretically aligned into membrane-membrane contact with a weak alternating electric field as much as 5 min after ... More
In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins.
AuthorsNewmeyer DD, Finlay DR, Forbes DJ
JournalJ Cell Biol
PubMed ID3097026
An in vitro system was developed that provides a quick microscopic assay for nuclear transport. The assay uses an extract of Xenopus eggs, normal or synthetic nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. This in vitro system accurately mimics in vivo nuclear transport, both in exclusivity and in the ... More