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Biomedical subjects

I Giaever

Publications and source records attributed to I Giaever.

At least 37 records · Page 2Linked to original sources

Monitoring electropermeabilization in the plasma membrane of adherent mammalian cells.

When an electrical potential of order one volt is induced across a cell membrane for a fraction of a second, temporary breakdown of ordinary membrane functions may occur. One result of such a breakdown is that molecules normally excluded by the membrane can now enter the cells. This phenomenon, generally referred to as electropermeabilization, is known as electroporation when actual pores form in the membrane. This paper presents a unique approach to the measurement of pore formation and closure in anchored mammalian cells. The cells are cultured on small gold electrodes, and by constantly monitoring the impedance of the electrode with a low-amplitude AC signal, small changes in cell morphology, cell motion, and membrane resistance can be detected. Because the active electrode is small, the application of a few volts across the cell-covered electrode causes pore formation in the cell membrane. In addition, the heat transfer is very efficient, and the cells can be porated in their regular growth medium. By this method, the formation and resealing of pores due to applied electric fields can be followed in real time for anchorage-dependent cells.

Biophysical Phenomena↗

Electrical resistance method for measuring volume changes in monolayer cultures applied to primary astrocyte cultures.

An electrical resistance method was developed to measure volume changes in substratum-attached monolayer cultures. Astrocytes in primary monolayer cultures prepared from neonatal rat cerebral cortex were placed in a confined channel containing a balanced salt solution, and the electrical resistance of the channel was measured using an applied alternating current. If the volume of the cells increases, then the volume of the solution within the channel available for current flow decreases by the same amount, resulting in an increase in the measured resistance through the channel. If the volume of the cells decreases, a decrease in resistance would be recorded. This method allows continuous measurements of volume changes in real time. When primary astrocyte monolayers were exposed to hyposmotic solutions (93-193 mosmol/kgH2O), they showed a rapid initial swelling and, in the continued presence of hyposmotic media, a characteristic regulatory volume decrease (RVD) in which there was a return to normal cell volume within approximately 20 min. Astrocytes exposed to hyperosmotic media (343-493 mosmol/kgH2O) gave a decrease in electrical resistance, indicating shrinkage. Putative endogenous effectors of astrocytic swelling, such as high extracellular K+ and glutamate, resulted in a much slower onset of swelling and no sign of RVD. This system can reliably measure the average change in cell monolayer volume to 1-2% and thus provides a sensitive means of continuous measurements of changes in cell volume in monolayer cultures.

Animals↗

Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function.

We have developed an electrical method to study endothelial cell shape changes in real time in order to examine the mechanisms of alterations in the endothelial barrier function. Endothelial shape changes were quantified by using a monolayer of endothelial cells grown on a small (10(-3) cm2) evaporated gold electrode and measuring the changes in electrical impedance. Bovine pulmonary microvessel endothelial cells and bovine pulmonary artery endothelial cells were used to study the effects of alpha-thrombin on cell-shape dynamics by the impedance measurement. alpha-Thrombin produced a dose-dependent decrease in impedance that occurred within 0.5 min in both cell types, indicative of retraction of endothelial cells and widening of interendothelial junctions because of "rounding up" of the cells. The alpha-thrombin-induced decrease in impedance persisted for approximately 2 hr, after which the value recovered to basal levels. Pretreatment of endothelial cells with the protein kinase C inhibitor, calphostin C, or with 8-bromoadenosine 3',5'-cyclic monophosphate prevented the decreased impedance, suggesting that the endothelial cell change is modulated by activation of second-messenger pathways. The alpha-thrombin-induced decrease in impedance was in agreement with the previously observed increases in transendothelial albumin permeability and evidence of formation of intercellular gaps after alpha-thrombin challenge. The impedance measurement may be a valuable in vitro method for the assessment of mechanisms of decreased endothelial barrier function occurring with inflammatory mediators. Since the rapidly occurring changes in endothelial cell shape in response to mediators such as thrombin are mediated activation of second-messenger pathways, the ability to monitor endothelial cell dynamics in real time may provide insights into the signal-transduction events mediating the increased endothelial permeability.

Animals↗

Micromotion of mammalian cells measured electrically.

Motility is a fundamental property of mammalian cells that normally is observed in tissue culture by time lapse microscopy where resolution is limited by the wavelength of light. This paper examines a powerful electrical technique by which cell motion is quantitatively measured at the nanometer level. In this method, the cells are cultured on small evaporated gold electrodes carrying weak ac currents. A large change in the measured electrical impedance of the electrodes is observed when cells attach and spread on these electrodes. When the impedance is tracked as a function of time, fluctuations are observed that are a direct measure of cell motion. Surprisingly, these fluctuations continue even when the cell layer becomes confluent. By comparing the measured impedance with a theoretical model, it is clear that under these circumstances the average motions of the cell layer of 1 nm can be inferred from the measurements. We refer to this aspect of cell motility as micromotion.

Cell Line↗

Substrate mechanics and cell spreading.

Cell spreading and cell locomotion arise from forces exerted by actin microfilaments upon the substratum. Using modified protein films at fluorocarbon oil--water interfaces as substrates, we have measured some minimal mechanical properties required of these films to support cell spreading forces in vitro. For murine 3T3-L1 fibroblasts, complete cell spreading was obtained when the films exceeded surface shear moduli and surface fracture points of 15 and 5 dyne/cm, respectively. The human WI-38 fibroblast required more robust films than did its transformed counterpart (WI-38/VA 13) in order to achieve equivalent spreading. These results are of significance in understanding the metastatic capabilities of cancer cells.

Actin Cytoskeleton↗

Electric measurements can be used to monitor the attachment and spreading of cells in tissue culture.

A new electrical assay to measure the attachment and spreading of cells in tissue culture has been developed and substantiated by comparison with a more conventional assay. Small gold electrodes are vacuum deposited on the bottom of standard polystyrene culture dishes and coated with various proteins. As mammalian fibroblasts attach and spread on these surfaces, the measured impedance of the electrodes changes. These impedance changes reflect the amount of area blocked by the spreading cells. Since the weak electrical signals used have no noticeable effects on the cells, this is a very convenient method that is both quantitative and sensitive for measuring cell attachment and spreading.

Animals↗

Measurement of macrophage adherence and spreading with weak electric fields.

A new method to monitor macrophage attachment on protein-coated surfaces and spreading in response to activating agents is described. Murine macrophages were cultured on small gold electrodes coated with protein, and attachment and spreading were detected as electrical impedance changes. The rate of attachment of cells to fibronectin-coated electrodes was measured to be significantly greater than to other proteins tested. Activation agents used included interferon-gamma, lipopolysaccharide and heat killed Listeria monocytogenes. Addition of each agent to macrophages on electrodes resulted in characteristic patterns in the impedance time course with impedance changes as large as 40%.

Animals↗

A screening technique for monoclonal antibody production: application of an indium slide immunoassay.

A simple technique is described that is suitable for rapid screening of hybridoma microculture fluids for monoclonal antibody producing hybrids. The procedure measures increase in light scatter due to the antigen-antibody reaction on a surface of indium metal coated upon glass and does not require use of a labeled second antibody. Techniques minimizing nonspecific binding in such assays are presented. The procedure was used to screen hybridoma microculture fluids containing mouse monoclonal antibodies directed against human mitochondrial isoenzyme of aspartate aminotransferase (EC 2.6.1.1). The technique was compared to an enzyme-linked immunosorbent assay, and similar semi-quantitative results were found for sample cultures tested in the two procedures. The new screening procedure affords a simple screening assay without sacrificing sensitivity or specificity of standard methods.

Animals↗

An agglutination assay using emulsified oils.

Dispersed fluorocarbon oil droplets were used as the antigen-carrier particle in an immunological agglutination assay. The extent of agglutination was found to vary with the percentage of antigen to that of inert protein used in the solution to coat and stabilize the oil droplets. Maximum sensitivity was observed when 6.25% of the protein adsorbed on the oil droplets was specific antigen. At this percentage, antibodies were detected at concentrations as low as 0.10 micrograms/ml. Results were obtained by visual examination of the reacted samples as well as with quantitative computer analysis using the Zeiss IBAS image analysis system.

Agglutination Tests↗

Monitoring fibroblast behavior in tissue culture with an applied electric field.

Mammalian fibroblasts have been cultured on evaporated gold electrodes subjected to an alternating electric field at 4000 Hz. The system consists of a large (approximately equal to 2 cm2) and a small (approximately equal to 3 X 10(-4) cm2) electrode bathed in tissue culture medium. The applied electric field produces a voltage drop at the boundary between the solution and the small electrode of a few mV at a current density of a few mA/cm2. The small population of cells that attach and spread on this electrode have a marked effect on the measured impedance and also cause it to fluctuate with time. The amplitude of these fluctuations is greatly reduced by cytochalasin B (10 microM), suggesting they are a consequence of cell movement.

Animals↗

A new assay for rheumatoid factor.

This solid-phase immunoassay for detection of rheumatoid factor involves a specially prepared indium surface that renders protein layers visible and therefore does not require tagged protein. Using appropriate discriminatory values, we found agreement with results of a standard latex-fixation test in controls and patients with rheumatoid arthritis. About half of the patients with rheumatoid arthritis that were seronegative by this latex fixation test had positive tests by the present assay. Rheumatoid factor of both the IgM and IgA class may be easily assessed.

Arthritis, Rheumatoid↗

Cell growth on liquid microcarriers.

Anchorage-dependent cell growth is demonstrated on microcarriers of fluorocarbon fluid formed by emulsification and stabilized with polylysine.

Animals↗

Behavior of cells at fluid interfaces.

We have cultured the murine cell lines 3T3-L1 and SV-T2 using as a substrate the layer of denatured protein that forms at the phase boundary between culture medium and fluorocarbon fluids. The growth patterns observed on these interfaces differ from those seen on conventional solid substrates. Depending on the cell strain and the composition of the fluorocarbon fluid, cells will tend to clump into isolated aggregates or form nearly confluent cell monolayers containing "lake-like" openings. We demonstrate that these growth patterns can be attributed to the ability of cultured cells to stress and break the protein monolayer on which they grow.

Animals↗

Cell growth on liquid interfaces: Role of surface active compounds.

Anchorage-dependent fibroblasts can be cultured by using as a substrate the protein layer that spontaneously forms at the liquid-liquid interface between fluorocarbon fluids and tissue culture medium. For this novel substrate to be effective in supporting confluent cell layers, the protein monolayer must support the stresses exerted by spreading fibroblasts. The composition of the fluorocarbon fluid has a significant effect on the strength of the protein layer and, thus, on the patterns of cell growth. Evidence is presented demonstrating that a protein film, sufficiently strong to support cell growth, does not occur on purified fluorocarbon fluids but requires the presence of trace amounts of polar, surface active compounds. By the addition of small quantities of pentafluorobenzoyl chloride to alumina-treated fluorocarbon fluids, excellent interfacial substrates can be produced. We have applied this understanding to produce a liquid microcarrier system capable of general use with a variety of cells, including human fibroblasts. A microcarrier in which the fluorocarbon is replaced with polydimethyldiphenyl siloxane is also described.

Journal Article↗

Measurement of antigen concentrations with a one-step inhibition assay.

A device is described which is used to measure the concentration of antigen in solution. A wedge-shaped gap containing the antigen to be detected is bounded on one side by a reservoir containing specific antiserum and on the opposite side by a detection slide which has been previously coated with a layer of adsorbed antigen. As the antibodies diffuse through the gap, they complex with the soluble antigen in the wedge until these become saturated leaving additional antibody free to complex with antigen on the detection slide. By allowing diffusion to continue for an appropriate time, it is possible to relate the distance along the gap where this has occurred to the concentration of antigen within the gap. Using human IgG as a model antigen, we have been able to measure concentrations down to approximately 10 micrograms/ml with this test.

Animals↗

Direct immunological determination of aspartate aminotransferase isoenzymes.

We examined the suitability of a rapid immunological technique to determine the amount of aspartate aminotransferase (EC 2.6.1.1) isoenzymes in human serum or tissue extracts. Purified isoenzymes were absorbed as a monolayer to the surface of an indium metal film on glass. The enzyme retains immunological reactivity, allowing the specific binding of aspartate aminotransferase antibodies at the surface. The amount of isoenzyme in a specimen is estimated from the competition for the antibody between the free isoenzyme in the specimen and that at the surface. The surface is further incubated with goat antibodies to rabbit IgG, and the extent of antibody binding is determined by densitometry. There is no cross reactivity between the cytoplasmic and mitochondrial forms, so these two isoenzymes can be determined simultaneously. The minimum detectable concentration by this technique is about 50 micrograms of enzyme protein per liter. The within-day coefficient of variation for determination of either isoenzyme was about 20%. Our results suggest that normal and patients' sera contain considerably more immunologically active than catalytically active isoenzymes.

Aspartate Aminotransferases↗

A variation of the radioimmune assay.

A novel method of radioimmune assay is described. It departs from conventional assays in that the radioiodine is reacted with protein following, rather than before, the antigen-antibody reaction. Using a BSA/anti-BSA system, the assay can detect antigen concentrations down to approximately 100 ng/ml.

Antibodies↗