Biomedical subjects

M A Model

Publications and source records attributed to M A Model.

10 recordsLinked to original sources

PubMed2001

A standard for calibration and shading correction of a fluorescence microscope.

BACKGROUND: Numerous applications of fluorescence microscopy require quantitation of signal intensity in reproducible units. Two problems must be overcome to achieve this goal. First, due to various instrumental factors, the same sample imaged on two microscopes or even on the same microscope at different times may produce highly divergent readings. Second, because of shading, some areas within the same field may appear brighter than others despite the same amount of fluorophore. The first type of variability requires calibration using a sample of reproducible fluorescence yield; to correct for shading, a uniform fluorescent field is needed. METHODS: Standard slides were prepared by placing several microliters of 10%-50% w/v fluorescein or rhodamine between a coverglass and a slide. They were used to perform shading correction and normalization under a variety of imaging conditions. RESULTS: Concentrated fluorophores produced a uniform fluorescent field of moderate and reproducible brightness. By expressing the staining of a biological object in the units of standard slides, identical results were obtained irrespective of the imaging conditions or the microscope used. We compared shading correction based on concentrated fluorescein with two other standards. Concentrated fluorescein resulted in the best equalization of the field. CONCLUSIONS: Standardization of fluorescent images can be achieved by normalizing them to the image of a concentrated solution of a fluorophore. Due to its simplicity and efficiency, this method can be used in clinical analysis as well as in routine laboratory practice.

Calibration↗

PubMed2000

Quantification of the surface density of a fluorescent label with the optical microscope.

Fluorescence microscopy can offer unique advantages for biomaterials characterization. Like spectroscopy or radioactivity, it can be used to quantify specific binding to surfaces, but it can also assess surface homogeneity at the micron scale or detect protein aggregation. To fully utilize the potential of this technique, there must be a way to calibrate the microscope in terms of the moles of a fluorophore per unit area. The method we propose involves the following steps: fluorescent labeling of erythrocytes and quantification of the label by flow cytometry; flattening of fluorescent erythrocytes for microscopic observation; imaging and digital analysis to relate the gray level intensities to the fluorophore density; and using this procedure to characterize a different, more easily obtainable, standard. The latter can be a 50% solution of Na fluorescein that yields a highly reproducible and uniform fluorescence. Concentrated fluorescein solution can also be used to correct images for the spatial nonuniformity of illumination and detection (shading correction). By applying this method to study the binding of IgG and fibrinogen to glass or amidated glass, we showed that protein adsorption to glass may result in protein aggregation that may affect the biological activity of the adsorbed protein.

Adsorption↗

PubMed1999

Optimization of the cost and sensitivity of receptor- and enzyme-based assays.

In detecting receptor antagonists or enzyme inhibitors, there are three parameters that often affect the outcome in a predictable quantitative manner: concentrations of the receptors (enzyme), labeled ligand (substrate), and antagonist (inhibitor). The usual goal of assay optimization is to maximize the ability of the assay to detect low concentrations of the analyte. Another question of practical importance, especially in screening of large numbers of samples, would be minimization of the reagent cost. Although the mathematical theory of optimization of the receptor binding assay was developed a long time ago, the resulting formulas (in the general case of unequal affinities of ligand and competitor) were not well suited for practical use. The current availability of computational programs, such as Mathematica, makes possible an efficient solution, both for receptor- and enzyme-based assays. We use a graphical approach to assay optimization and apply it to the following problems: (1) optimization of assay sensitivity, (2) optimization of the reagent cost, and (3) analysis of the entire range of the parameter values since the mathematically optimal values may sometimes be impractical. The computation is extremely simple and the problem can sometimes be solved in several minutes.

Binding, Competitive↗

PubMed1998

Experimental approaches for observing homologous desensitisation and their pitfalls.

Whereas molecular mechanisms of cell desensitisation have been discussed at length in the literature, little organised information on the methods for studying desensitisation of cellular responses has been published. In this article, three commonly utilised protocols for studying homologous desensitisation of cellular responses are evaluated. These are (1) observation of attenuation of a response after an initial stimulus-induced activation, (2) repeated stimulation of cells after washing away the previous stimulus, and (3) repeated stimulation without a wash step. Advantages and limitations of each protocol are discussed and data is presented demonstrating some of the properties of the protocols.

Animals↗

PubMed1998

Experimental approaches for observing homologous desensitisation and their pitfalls.

Whereas molecular mechanisms of cell desensitisation have been discussed at length in the literature, little organised information on the methods for studying desensitisation of cellular responses have been published. In this article, three commonly utilised protocols for studying homologous desensitisation of cellular responses are evaluated. These are (1) observation of attenuation of a response after an initial stimulus-induced activation, (2) repeated stimulation of cells after washing away the previous stimulus, and (3) repeated stimulation without a wash step. Advantages and limitations of each protocol are discussed and data is presented demonstrating some of the properties of the protocols.

Cell Communication↗

PubMed1998

A microscopic study of Fc gamma RIII-mediated respiratory burst in neutrophils.

We investigated the relationship between the respiratory burst in neutrophils and the membrane distribution of the IgG receptor, Fc gamma RIII. Fc gamma RIII receptors were labeled with a fluoresceinated antibody that does not block binding of immune complexes. The respiratory burst was detected using covalently bound rosamine stain previously described for flow cytometric applications. This method allows visualization of intracellular oxidant production in fixed cells using attenuated illumination with a laser. Strong cytosolic oxidation of rosamine was observed only in those cells that displayed prominent receptor endocytosis upon interaction with insoluble immune complexes. Soluble immune complexes or insoluble complexes in the presence of cytochalasin B did not stimulate endocytosis of Fc gamma RIII and induced no rosamine oxidation. Extracellular superoxide production measured by the cytochrome c test did not correlate with intracellular rosamine oxidation: it was maximal in cytochalasin-treated cells and did not require any visible receptor rearrangement. Our results demonstrate the utility of the rosamine stain as an intracellular marker of the oxidative burst, support the role of Fc gamma RIII in neutrophil activation and emphasize the compartmental regulation of the oxidative burst.

Antigen-Antibody Complex↗

PubMed1997

A sensitive flow cytometric method for measuring the oxidative burst.

We report a novel method of flow cytometric detection of the oxidative burst in human neutrophils. The cells were covalently labeled on the plasma membrane by incubation with 4-carboxydihydrotetramethylrosamine succinimidyl ester on ice. Activation of neutrophils resulted in an increase in red fluorescence at 575 nm using 514 nm excitation. The sensitivity of this assay in detecting the response to chemotactic peptide N-formyl-met-leu-phe (FMLP) and phorbol 12-myristate 13-acetate (PMA) was comparable to that observed with the cytochrome c oxidation test. We compared the new method to previously described methods using intracellular fluorescent stains. Our method showed the lowest nonspecific oxidation and the best sensitivity to FMLP-induced activation. It was equally or slightly less efficient than the green fluorescent stain dihydrorhodamine 123 in detection of neutrophil activation by immune complexes and PMA but much more sensitive than red fluorescent stains hydroethidine and dihydrotetramethylrosamine. It can be successfully used in kinetic experiments and yields a fluorescence signal that remains stable for at least 1.5 h after formaldehyde fixation with antioxidant added.

Flow Cytometry↗

PubMed1996

Cell polarization as a possible mechanism of response termination.

Neutrophils stimulated with a low concentration of chemotactic peptide N-formyl-met-leu-phe respond with a brief pulse of actin polymerization and a persistent change in morphology from round to polarized. The recovery of the actin polymerization response is unlikely to be due to usual receptor desensitization mechanisms. We hypothesized that cell polarization and redistribution of signaling components effect the depolymerization phase of the actin response. In this report we show that the overall actin depolymerization is equivalent to the reduction in the volume occupied by F-actin while its concentration at the front of the polarized cell remains undiminished. To test whether the confinement of F-actin to a small volume can be caused by receptor redistribution, we observed receptors and F-actin in the same cell and found that their localization was different. To explain our findings, we propose a model based on the affinity of a signaling component, other than the receptor, for the areas of the cell rich in F-actin.

Actins↗

PubMed1995

Desensitization of the actin polymerization response in human neutrophils at low cell density.

Many chemoattractant-activated responses in neutrophils show transient kinetics, suggesting that rapid desensitization occurs during the time course of the response. We found that desensitization of the actin polymerization response to N-formyl peptides is, in a large part, due to inhibition by adenosine released from cells to the medium and depletion or a chemical inactivation of the agonist. To reduce the influence of these factors, we stimulated neutrophils in a very diluted suspension, sometimes with continuous replacement of the medium. The actin polymerization response to a high agonist concentration was greatly enhanced and prolonged under these conditions, often without any tendency to subside within 10 min at 25 degrees C. It has previously been shown that the N-formyl peptide receptor converts from a rapidly dissociating to a slowly dissociating and presumably inactive form during activation. Under the conditions of low cell concentration, the conversion to a slowly dissociating receptor still occurred. Thus the prolonged response was not due to prolonged presence of rapidly dissociating receptors. We conclude either that a low number of rapidly dissociating receptors, which we failed to see, is sufficient to maintain actin polymerization or that slowly dissociating receptors can support the actin response. In contrast to responses stimulated by high agonist concentrations, the responses to low concentrations of the agonists were transient. The results of other authors indicate that low concentrations of N-formyl peptides do not desensitize the receptors. Other mechanisms, which are specific for the actin polymerization response, must be involved in response termination to low concentrations of N-formyl peptides. Activation at low cell density will be a useful approach for studying other processes (Ca2+ elevation, oxidant production, etc.) and chemoattractants (leukotriene B4, interleukin 8, etc.) for which an understanding of the kinetics due to desensitization of the components of the receptor-mediated activation pathway is desired.

Actin Cytoskeleton↗

PubMed1995

Ligand-receptor interaction rates in the presence of convective mass transport.

The rate of binding of a ligand to receptors on the cell surface can be diffusion limited. We analyze the kinetics of binding, diffusion-limited in a stationary liquid, in the presence of convective mass transport. We derive a formula that expresses the reaction kinetics in terms of the mass transfer coefficient. A moderately transport-limited kinetics is not readily recognizable from the shape of the binding curve and may lead to erroneous estimates of the rate coefficients. We apply our results to practically important cases: a cell suspension in a stirred volume of liquid and a confluent cell colony under a laminar stream. Using typical numbers characterizing the ligand-receptor interactions, we show that stirring and perfusion can be important factors determining the reaction rates. With the confluent colony, the early reaction kinetics requires a different treatment, and we provide it for the case of low receptor occupancy. We show that, even with a fast perfusion, a cell monolayer can transiently generate a zone of depletion of the ligand, and that would affect the early stages of the reaction. Our results are expressed in a simple analytical form and can be used for the design and interpretation of experimental data.

Animals↗