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Biomedical subjects

L A Ernst

Publications and source records attributed to L A Ernst.

9 recordsLinked to original sources
PubMed1992

Flow cytometric analysis of lymphocyte phenotypes in bronchoalveolar lavage fluid: comparison of a two-color technique with a standard immunoperoxidase assay.

Characterization of lymphocytes in bronchoalveolar fluid has provided insight into the pathogenesis of many pulmonary diseases. Identification of lymphocyte phenotypes has become highly successful due to development of specific monoclonal antibodies and reliable methods for detecting labeled cells such as flow cytometry (FCM) and immunocytochemistry. FCM permits rapid screening of many cells, but this analysis may be confounded by heterogeneity in the size and granularity of the cells being evaluated. Such heterogeneity may lead to exclusion of cells of interest and inclusion of unwanted cells. Often peripheral blood leukocytes are used to define the gate for lung lymphocytes, but this gate may be inappropriate due to considerable variation in size and granularity of cells in bronchoalveolar lavage (BAL) fluid. Here we report an alternative method for generating a gate which employed fluorescence and side scatter signals to analyze lymphocyte subsets in BAL fluid by FCM. This gating technique avoids the pitfalls inherent in using the conventional lymphocyte gate to analyze lung cells. To validate this approach, we compared the results generated by this gate and those from the conventional forward/side light scatter gate to results derived from an immunocytochemical technique (ABC) that has been extensively employed in our laboratory to identify lymphocyte subsets in blood and lavage fluid. FCM tended to underestimate the proportions of T-cell subsets compared with ABC when the conventional gate was used. Counting only cells that stained with fluorescein-conjugated anti-CD45 antibody and that had side scatter properties of lymphocytes, however, resulted in excellent agreement between FCM and ABC. It appears that the CD45+/side scatter gate includes the vast majority of lymphocytes in BAL fluid while excluding most of the nonlymphoid cells that contaminate the conventional gate. It was this latter group of cells, and erythrocytes in particular, that led to the artificially low values for lymphocyte phenotypes in BAL fluid by FCM when the conventional lymphocyte gate was used. Although erythrocytes in BAL fluid may be eliminated by hypotonic lysis, this may also result in contamination of the conventional lymphocyte gate with nuclear debris and particulates from macrophages. Despite these advantages, the fluorescence/side scatter gate may not always be optimal for the evaluation of T lymphocytes if BAL fluid contains CD45+, nonlymphoid cells with low side light scatter. In these instances, additional antibodies such as anti-CD14 and anti-CD11 may be employed to determine the size of contaminant monocytic cells and neutrophils.(ABSTRACT TRUNCATED AT 400 WORDS)

Bronchoalveolar Lavage Fluid↗
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

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

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↗
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

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↗