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A S Waggoner

Publications and source records attributed to A S Waggoner.

At least 19 recordsLinked to original sources
PubMed1991

Imaging cytometry by multiparameter fluorescence.

A system is described for performing multicolor fluorescence image cytometry of cell preparations. After the setting up stage, the operation is automatic: the microscope fields are found and focused; then images are acquired for each fluorophore, corrected and analyzed, without any operator interaction. Human peripheral blood lymphocytes on microscope slides were used as a test system. In these experiments, three fluorescent antibodies were used to identify lymphocyte sub-populations, and a DNA content probe was used to identify all nucleated cells. The cell subset percentages determined by image cytometry were comparable to percentages obtained when cells from the same preparation were analyzed by flow cytometry. Multicolor fluorescence imaging cytometry can potentially be extended to the analysis of cells in smears, fine needle biopsies, imprints, and tissue sections.

Algorithms↗
PubMed1990

A calcium-sensitive fluorescent analog of calmodulin based on a novel calmodulin-binding fluorophore.

Structure-activity studies of tetramethinemerocyanine fluorophores enabled the synthesis of novel dyes which showed spectral changes during reversible, calcium-dependent association with calmodulin. These spectral changes were greatly enhanced in dyes with a quaternary nitrogen and specifically placed hydrophobic chains. One such dye was covalently attached to calmodulin, producing a calmodulin analog with calcium-sensitive fluorescence. The analog, MeroCaM, showed a calcium-induced 3.4-fold increase in excitation ratio (608/532 nm excitation, 623 nm emission), which was fully reversed by lowering free calcium levels. MeroCaM's excitation ratio showed a half-maximal change at 300-400 nM calcium, below calcium concentrations reported to produce half-maximal saturation of calcium-calmodulin binding. However, the calcium dependence of MeroCaM's phosphodiesterase activation paralleled that of calmodulin. MeroCaM's fluorescence changes therefore appear to reflect primarily calcium binding to high affinity sites. MeroCaM's maximal phosphodiesterase activation was 30-40% that of calmodulin. In myosin light chain kinase activation, MeroCaM and calmodulin displayed indistinguishable maximal activation levels and concentration dependence of activation. Changes in MeroCaM's calcium affinity induced by magnesium, phosphodiesterase, and melittin were similar to those reported for calmodulin. Experiments with melittin revealed that target protein interaction could alter the fluorescence changes produced by calcium binding. MeroCaM showed promising brightness and photostability when imaged in individual living fibroblasts. The long excitation and emission wavelengths of MeroCaM, and the strong dependence of its excitation ratio on calcium concentrations, suit it well for use as a probe of calmodulin-dependent calcium signaling in living cells, as well as for experiments in vitro.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1990

Cyanine dye labeling reagents--carboxymethylindocyanine succinimidyl esters.

Ten carboxymethylindocyanine dyes which form the basis of a new series of fluorescent probes have been synthesized and converted into succinimidyl active esters for fluorescent labeling of proteins or other amino-containing substances. Fluorescence emission maxima for members of the series range from 575 to 780 nm. Hydrophilic, water-soluble reagents have been obtained which yield labeled antibodies with little tendency to form precipitates. The fluorescence intensities achieved are higher than those produced by labeling with the cyanine isothiocyanates described previously (Mujumdar et al.: Cytometry 10:11-19, 1989). The utility of these reagents has been demonstrated in antibody labeling for two-color immunofluorescent imaging of internal structures in a mammalian cell and for two-color flow-cytometry experiments. The use of values of chromophore-equivalent weight (W/Ceq), calculated from quantitative absorption data on dye samples, is proposed as an aid in formulating labeling procedures.

Animals↗
PubMed1989

Use of 4-heptylumbelliferyl-beta-D-glucoside to identify Gaucher's disease heterozygotes.

Three fluorometric beta-glucosidase assays were compared for their ability to identify Gaucher's disease heterozygotes, using leukocytes as the source of enzyme: the pH 5.5-taurocholate assay of Peters et al.; the conduritol B epoxide dependent variation of that assay; and the newly developed method described herein. While the first two procedures utilize the standard substrate 4-methylumbelliferyl-beta-D-glucopyranoside to estimate beta-glucosidase activity, the new assay uses 4-heptylumbelliferyl-beta-D-glucoside as (C7UGlc) substrate. Use of this substrate enhances the specificity of the method for lysosomal glucocerebrosidase, thereby minimizing the contribution of the nonspecific cytosolic beta-glucosidase to estimates of substrate hydrolysis. Using Student's t test for the three assays examined, the C7UGlc assay procedure was determined to have the lowest p value (p less than 0.001) and highest t value (t = 4.95) for the discrimination between the mean glucocerebrosidase value of control and obligate Gaucher heterozygote samples. The high reliability and simplicity of the C7UGlc assay lends adequate reason to favor this assay for regular clinical diagnosis of Gaucher heterozygotes.

Brain↗
PubMed1989

Cyanine dye labeling reagents for sulfhydryl groups.

Cyanine and merocyanine dyes are introduced as new fluorescent reagents for covalently labeling proteins and other biomolecules. These dyes, which contain iodoacetamide functional groups, have high extinction coefficients and moderate quantum yields. A major advantage of these polymethine dyes is the easy manipulation of their spectral properties during synthesis. Cyanines containing reactive functional groups can be made with absorption maxima ranging from less than 500 nm to greater than 750 nm. This property opens additional regions of the spectrum for experiments involving the simultaneous multicolor analysis of different fluorescent probes. The cyanines, which are relatively insensitive to solvent property changes, are complemented by the merocyanines, which are keen indicators of solvent polarity.

Carbocyanines↗
PubMed1989

Cyanine dye labeling reagents containing isothiocyanate groups.

New isothiocyanate derivatives of cyanine dyes were synthesized as fluorescent covalent labeling reagents for proteins and other biomolecules. These dyes have maximum absorbance in the red and near infrared regions of the spectrum, have high extinction coefficients and have adequate quantum yields. Incorporating two alkyl sulfonate groups in the dye structures increases their water solubility, which is beneficial for labeling biological molecules in aqueous solution. Reactivities of proteins with these new cyanines are similar to their reactivities with fluorescein isothiocyanate. These new labeling reagents are complementary to the fluorescein and rhodamine reagents, expanding the possibilities of multicolor analyses. Sheep anti-mouse-IgG antibody was labeled with a pentamethine cyanine dye (CY5.8-ITC) and used with a fluoresceinated antibody as a second reagent for detecting human T-cell subsets by flow cytometry.

Antibodies↗
PubMed1989

Calcium signals recorded from two new purpurate indicators inside frog cut twitch fibers.

Two new Ca indicators, purpurate-3,3'diacetic acid (PDAA) and 1,1'-dimethylpurpurate-3,3'diacetic acid (DMPDAA), were synthesized and used to measure Ca transients in frog cut muscle fibers. These indicators are analogues of the purpurate components of murexide and tetramethylmurexide, in which two acetate groups have been incorporated into each molecule to render it membrane impermeant. The apparent dissociation constant for Ca is 0.95 mM for PDAA and 0.78 mM for DMPDAA. One of the indicators was introduced into a cut fiber, which was mounted in a double Vaseline-gap chamber, by diffusion from the end-pool solutions. The time course of indicator concentration, monitored optically in the middle of the fiber in the central-pool region, suggests that 19% of the PDAA or 27% of the DMPDAA became bound or sequestered inside the fiber. In resting fibers, the absorbance spectrum of either indicator was well fitted by the indicator's [Ca] = 0 mM cuvette absorbance spectrum, which is consistent with the idea that PDAA and DMPDAA do not enter the sarcoplasmic reticulum as tetramethylmurexide appears to be able to do (Maylie, J., M. Irving, N.L. Sizto, G. Boyarsky, and W. K. Chandler, 1987. Journal of General Physiology. 89:145-176). After an action potential, the absorbance of either indicator underwent a rapid and transient change that returned to the prestimulus baseline within 100-200 ms. The amplitude of this change had a wavelength dependence that matched the indicator's Ca-difference spectrum. The average amplitude of peak free [Ca] was 21 microM (PDAA or DMPDAA) if all the indicator inside a fiber was able to react with Ca as in cuvette calibrations, and was 26 (PDAA) or 28 microM (DMPDAA) if only freely diffusible indicator could so react. These results suggest that PDAA and DMPDAA are the first Ca indicators that provide a reliable estimate of both the amplitude and time course of (the spatial average of) free [Ca] in a twitch muscle fiber after an action potential.

Action Potentials↗
PubMed1988

Impermeant potential-sensitive oxonol dyes: II. The dependence of the absorption signal on the length of alkyl substituents attached to the dye.

We have measured the potential-dependent light absorption changes of 43 impermeant oxonol dyes with an oxidized cholesterol bilayer lipid membrane system. The size of the signal is strongly dependent on the chain length of alkyl groups attached to the chromophore. Dye molecules with intermediate chain lengths give the largest signals. To better understand the dependence of the absorbance signal on alkyl chain length, a simple equilibrium thermodynamic analysis has been derived. The analysis uses the free energy of dye binding to the membrane and the "on-off" model (E.B. George et al., J. Membrane Biol., 103:245-253, 1988a) for the potential-sensing mechanism. In this model, a population of dye molecules in nonpolar membrane binding sites is in a potential-dependent equilibrium with a second population of dye that resides in an unstirred layer adjacent to the membrane. Dye in the unstirred layer is in a separate equilibrium with dye in the bulk bathing solution. The equilibrium binding theory predicts a "sigmoidally shaped" increase in signal with increasing alkyl chain length, even for very nonpolar dyes. We suggest that aggregation of the more hydrophobic dyes in the membrane bathing solution may be responsible for their low signals, which are not predicted by the theory.

Adsorption↗
PubMed1988

Impermeant potential-sensitive oxonol dyes: III. The dependence of the absorption signal on membrane potential.

We have measured potential-dependent changes in the absorption of light by oxidized cholesterol bilayer lipid membranes in the presence of impermeant oxonol dyes. The magnitude of the absorption signal increased linearly with the size of potential steps over a range of 500 mV. The signal also increased when the offset voltage of the pulse train was increased from -150 to +150 mV. The data are consistent with the "on-off" mechanism proposed by E. B. George et al. (J. Membrane Biol. 103:245-253, 1988) in which the probe undergoes potential-dependent movement between a binding site in the membrane and an aqueous region just off the surface of the membrane. An equilibrium thermodynamic analysis of the experimental data indicates that the negatively charged oxonol chromophore senses only 5-10% of the total membrane potential difference across the membrane when it is driven into a nonpolar binding site on the membrane.

Adsorption↗
PubMed1988

Impermeant potential-sensitive oxonol dyes: I. Evidence for an "on-off" mechanism.

This series of papers addresses the mechanism by which certain impermeant oxonol dyes respond to membrane-potential changes, denoted delta Em. Hemispherical oxidized cholesterol bilayer membranes provided a controlled model membrane system for determining the dependence of the light absorption signal from the dye on parameters such as the wavelength and polarization of the light illuminating the membrane, the structure of the dye, and delta Em. This paper is concerned with the determination and analysis of absorption spectral changes of the dye RGA461 during trains of step changes of Em. The wavelength dependence of the absorption signal is consistent with an "on-off" mechanism in which dye molecules are driven by potential changes between an aqueous region just off the membrane and a relatively nonpolar binding site on the membrane. Polarization data indicate that dye molecules in the membrane site tend to orient with the long axis of the chromophore perpendicular to the surface of the membrane. Experiments with hyperpolarized human red blood cells confirmed that the impermeant oxonols undergo a potential-dependent partition between the membrane and the bathing medium.

Benzenesulfonates↗
PubMed1987

Five-parameter fluorescence imaging: wound healing of living Swiss 3T3 cells.

Cellular functions involve the temporal and spatial interplay of ions, metabolites, macromolecules, and organelles. To define the mechanisms responsible for completing cellular functions, we used methods that can yield both temporal and spatial information on multiple physiological parameters and chemical components in the same cell. We demonstrated that the combined use of selected fluorescent probes, fluorescence microscopy, and imaging methods can yield information on at least five separate cellular parameters and components in the same living cell. Furthermore, the temporal and spatial dynamics of each of the parameters and/or components can be correlated with one or more of the others. Five parameters were investigated by spectrally isolating defined regions of the ultraviolet, visible, and near-infrared spectrum based on five distinct fluorescent probes. The parameters included nuclei (Hoechst 33342), mitochondria (diIC1-[5] ), endosomes (lissamine rhodamine B-dextran), actin (fluorescein), and the cell volume Cy7-dextran). Nonmotile, confluent Swiss 3T3 cells did not show any detectable polarity of cell shape, or distribution of nuclei, endosomes, or mitochondria. These cells also organized a large percentage of the actin into stress fibers. In contrast, cells migrating into an in vitro wound exhibited at least two stages of reorganization of organelles and cytoplasm. During the first 3 h after wounding, the cells along the edge of the wound assumed a polarized shape, carried the nuclei in the rear of the cells, excluded endosomes and mitochondria from the lamellipodia, and lost most of the highly organized stress fibers. The cell showed a dramatic change between 3 and 7 h after producing the wound. The cells became highly elongated and motile; both the endosomes and the mitochondria penetrated into the lamellipodia, while the nuclei remained in the rear and the actin remained in less organized structures. Defining the temporal and spatial dynamics and interplay of ions, contractile proteins, lipids, regulatory proteins, metabolites, and organelles should lead to an understanding of the molecular basis of cell migration, as well as other cellular functions.

Actins↗
PubMed1985

Light-scattering changes during chemotactic stimulation of human neutrophils: kinetics followed by flow cytometry.

The light-scattering properties of human neutrophils were compared on a cell-by-cell basis before and after stimulation with chemotactic peptide using flow cytometry. Between 20 and 180 sec after peptide addition, side (90 degrees) scatter declined by up to 4% and forward scatter increased up to 6%. Between 3 and 15 min, side scatter increased up to 15% and forward scatter decreased up to 5%. Association of a fluorescence chemoattractant with neutrophils was most rapid during the initial phase of increasing forward and decreasing side scatter, and association saturated before the maximum increase in side scatter. Evidence is presented that the observed changes in scatter were not a consequence of chemoattractant-induced cell-cell adhesion or neutrophil degranulation. Rather, the early phases of light-scattering changes are interpreted to represent membrane ruffling by the stimulated neutrophil; the later phases polarization of the neutrophil morphology.

Chemotaxis, Leukocyte↗
PubMed1985

Leishmania donovani: surface membrane acid phosphatase blocks neutrophil oxidative metabolite production.

We show that a purified preparation of the prominent tartrate-resistant acid phosphatase (E.C.3.1.3.2), isolated from the external surface of the intracellular parasite Leishmania donovani (promastigote form), inhibits toxic oxidative metabolite production of neutrophils. Preincubation of a neutrophil suspension (2.5 X 10(6) cells/ml) for 15 min at 37 C with 250 units (1 unit equals 1 nmole of 4-methylumbelliferyl phosphate cleaved per hr at pH 5.5) of the acid phosphatase in Krebs-Ringer phosphate buffer (pH 7.4) decreased O2 consumption, O2- production, and H2O2 production of N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated neutrophils to 15-25% of control values. The acid phosphatase also affected concanavalin A-stimulated O2-production by neutrophils, but had no effect on the rate of phorbol myristic acetate-stimulated O2- production, chemotactic peptide binding, degranulation, or membrane depolarization. Addition of an acid phosphatase inhibitor (Complex E; (NH4)6[P2Mo18O62] X 9H2O) to suspensions of opsonized promastigotes and neutrophils resulted in a threefold or greater enhancement of O2- production. These results suggest a possible pathophysiologic role for the acid phosphatase of L. donovani promastigotes.

Acid Phosphatase↗
PubMed1985

Structural organization of interphase 3T3 fibroblasts studied by total internal reflection fluorescence microscopy.

We studied the laminar organization of 3T3 fibroblast cells growing on glass slides by use of total internal reflection illumination to excite fluorescence emission (TIRF) from labeled molecules and stained cellular compartments that are very close to the cell-substrate contact region. Mitochondria, distant from the contact regions and stained with the water-soluble cationic dye, dil-C3-(3), fluoresced only as the glass/cytoplasm critical angle was approached. A similar result was obtained when the nuclei were stained with Hoechst dye 33342. From this measured angle a cytoplasmic refractive index in the range 1.358-1.374 was computed. The plasma membrane of 3T3 cells was stained with dil-C18-(3), and the cytoplasmic compartment was stained with fluoresceinyl-dextran (FTC-dextran) or with carboxyfluorescein. We have demonstrated a high degree of correspondence between the low-reflectance zones in the reflection interference image of a live cell and the TIRF images of both the plasma membrane and cytoplasmic compartment. TIRF photometry of selected contact regions of cells provided data from which the absolute separation of cell and substrate was computed. From a population of 3T3 cells microinjected with fluorescein-labeled actin, motile and adherent interphase cells were selected for study. For adherent cells, which displayed fluorescent stress fibers, the TIRF image was composed of intense patches and less intense regions that corresponded, respectively, to the focal contact and close-contact zones of the reflection-interference image. The intense patches corresponded to the endpoints of the stress fibers. Cells of motile morphology, which formed some focal contacts and extensive close-contact zones, gave AF-actin TIRF images of relatively even intensity. Thin lamellar regions of the cytoplasm were found to contain concentrations of actin not significantly different from other close-contact regions of the cell. The major analytical problem of TIRF microscopy is separation of the effects of proximity to substrate, refractive index, and fluorescent probe concentration on the local brightness of the TIRF image. From our results, it appears possible to use TIRF microscopy to measure the proximity of different components of substrate contact regions of cells.

Actins↗
PubMed1981

A cyanine dye distinguishes between cycling and non-cycling fibroblasts.

Cellular proliferative activity has previously been determined by measuring the incorporation of radiolabelled nucleotides or by visual inspection of cellular morphology. Although two flow cytometric methods have recently been developed which can distinguish cycling from non-cycling cells, both have serious disadvantages. One method requires uptake of a substantial amount of BUdR, limiting its usefulness for in vitro systems. The other method utilizes RNA/DNA content differences but its successful application has proved cell-type dependent. We have now used the findings that the cell membrane is more highly polarized in resting than in proliferating cells and that cyanine dyes carrying a delocalized positive charge enter live cells to an extent that depends on the cell membrane potential, to develop a method of distinguishing between cycling and non-cycling cells. The greater the membrane polarization, the greater is the concentration of dye within the cell. At high concentrations, the dye molecules aggregate and their fluorescence is quenched. Thus, for a given external dye concentration, cells of different membrane potential would accumulate different amounts of fluorescent (non-aggregated) dye. Using fibroblasts in culture conditions chosen to provide various models of cycling and non-cycling cells, we found that fluorescence intensity with the dye, 3,3'-diheptyloxycarbocyanine (Di-O-C,(3)) was consistently greater in the former than the latter.

Carbocyanines↗
PubMed1981

The importance of inorganic phosphate in regulation of energy metabolism of Streptococcus lactis.

This paper is concerned with the control of glycolysis in nongrowing Streptococcus lactis 7962. Changes were measured in the concentrations of glycolytic intermediates, intracellular inorganic phosphate (Pi), and adenine nucleotides following addition of glucose to cells that were in a starved condition. We find that intracellular Pi is a major factor in the control of glycolysis. In starved cells, the intracellular Pi concentration is high, greater than 40 mM. The large phosphoenolpyruvate pool that exists in starved cells can be explained as a result of inhibition of pyruvate kinase by the high concentration of Pi. On the other hand, in cells that are metabolizing glucose at a steady state rate, the cellular Pi concentration is low and pyruvate kinase is active. Upon depletion of glucose from the medium, the metabolite concentrations return to the values originally found in the starved state. This glucose depletion raises the intracellular Pi which again leads to inhibition of pyruvate kinase and the consequent buildup of the P-enolpyruvate pool.

Energy Metabolism↗