Micronuclei assay by laser scanning cytometry

BACKGROUND: The micronuclei (MN) assay is used to assess the chromosomal/mitotic spindle damage induced by ionizing radiation or mutagenic agents in vivo or in vitro. Because visual scoring of MN is cumbersome semi-automatic procedures that relay either on flow cytometry or image analysis were developed: both offer some advantages but also have shortcomings. METHODS: In the present study laser scanning cytometer (LSC), the instrument that combines analytical capabilities of flow and image cytometry, has been adapted for quantitative analysis of MN. The micronucleation of human breast carcinoma MCF-7 and leukemic HL-60 and U-937 cells was induced by in vitro treatment with mitomycin C. Cellular DNA was stained with propidium iodide (PI), protein was counterstained with fluorescein isothiocyanate (FITC). Two approaches were used to detect MN: (a) the threshold contour was set based on the data from the photosensor measuring red fluorescence of PI and MN were identified on the bivariate PI versus PI/FITC fluorescence distributions by their characteristic position; (b) the threshold contour was set on the data from the sensor measuring FITC fluorescence which made it possible, using the LSC software dedicated for FISH analysis, to assay both the frequency and DNA content of individual MN within each measured cell. RESULTS: The capability of LSC to relocate MN for visual examination was useful to confirm their identification. Visual identification of MN combined with their multiparameter characterization that took into an account their DNA content and protein/DNA ratio made it possible establish the gating parameters that excluded objects that were not MN; 93.3+/-3.3 events within the selected gate were MN. It was also possible to successfully apply FISH software to characterize individual cells with respect to quantity of MN residing in them. The percentage of MN assayed by LSC correlated well with that estimated visually by microscopy, both for MCF-7 (r = 0.93) and HL-60 cells (r = 0.87). CONCLUSIONS: LSC can be used to obtain unbiased estimate of MN frequencies. Unlike flow cytometry, it also allows one to characterize individual cells with respect to frequency and DNA content of MN residing in these cells. These analytical capabilities of LSC may be helpful not only to score MN but also to study mechanisms by which clastogenic agents induce MN.     

Quantification and functional analysis of chemotaxis by laser scanning cytometry.

BACKGROUND: Evaluation of chemotaxis assays traditionally relies on cumbersome and at times inaccurate visual counting. Moreover, many physiologic parameters that could be evaluated in conjunction with chemotactic migration, aside from morphologic changes, usually are not assessed due to the lack of a simultaneous method of analysis. We tested the suitability of laser scanning cytometry (LSC) as a convenient platform for counting migrated cells and for concurrent analysis of some features associated with their physiologic status. METHODS: We induced migration of THP-1 monocytes across Nuclepore filters with monocyte chemotactic protein-1 or vascular endothelial growth factor, alone or in combination. Filters were collected, and cells were fixed on filters and stained with the nuclear stain propidium iodide. Chemotactic indices were obtained by counting representative microscopic fields and by scanning the filters in LSC mode. RESULTS: We found an excellent correlation between direct counting and LSC. In addition, the software tools embodied in the LSC instrument allowed the observation of changes in nuclear compactness (increase in propidium iodide brightness) and morphology (increase in nuclear area and perimeter) that occurred in transmigrated cells. Monocyte chemotactic protein-1 and vascular endothelial growth factor acted as additive stimuli on these parameters. CONCLUSIONS: LSC analysis of cells undergoing chemotaxis provides a reliable and comprehensive assessment of the numbers and distribution of migrated cells and some of their nuclear parameters. The method can be easily extended to include the assessment of coincident molecular changes in cells due to chemotactic stimulation.     

Automation of mouse micronucleus genotoxicity assay by laser scanning cytometry

BACKGROUND: The evaluation of the safety of drugs and other chemicals is an important aspect of toxicology work. The mouse micronucleus assay is a standard in vivo genotoxicity assay. Chromosomal damage is an indicator of genotoxicity, which manifests in the formation of micronuclei in polychromatic erythrocytes from bone marrow and in peripheral blood erythrocytes. The assay is laborious to perform by manual counting. The laser scanning cytometer allows automated and rapid quantitation of cellular and subcellular fluorescence in monodisperse cell samples on a microscope slide. The object of this study was to evaluate the application of this new technology in the mouse micronucleus genotoxicity assay. Materials and Methods One hundred forty-four mice of various strains were dosed with combinations of carcinogens and antioxidants. Duplicate blood films were prepared 3 days later. One set of slides was stained with acridine orange, and the proportion of micronucleated erythrocytes was counted in 5,000 cells per slide. The duplicates were stained with propidium iodide (40 microg/ml). Five thousand cells per sample were examined using a laser scanning cytometer. The proportion of micronucleated erythrocytes was measured. RESULTS: A coefficient of correlation of 0.96 was found between the data from the two assays. The automation of the assay on the LSC produced a considerable time saving and efficiency gain. CONCLUSIONS: We conclude that with further development, laser scanning cytometry is likely to become the preferred modality for the performance of standard genotoxicity assays.     

Clinical applications of laser scanning cytometry

This study reviews existing and potential clinical applications of laser scanning cytometry (LSC) and outlines possible future developments. LSC provides a technology for solid phase cytometry. Fluorochrome-labeled specimens are immobilized on microscopic slides that are placed on a conventional epifluorescence microscope and analyzed by one or two lasers. Data comparable to flow cytometry are generated. In addition, the position of each event is recorded, a feature that allows relocalization and visualization of each measured event. The major advantage of LSC compared with other cytometric methods is the combination of two features: (a) the minimal clinical sample volume needed and (b) the connection of fluorescence data and morphological information for the measured event. Since the introduction of LSC, numerous methods have been established for the analysis of cells, cellular compartments, and tissues. Although most cytometric methods use only two or three colors, the characterization of specimens with up to five fluorochromes is possible. Most clinical applications have been designed to determine ploidy and immunophenotype; other applications include analyses of tissue biopsies and sections, fluorescence in situ hybridization, and the combination of vital and nonvital information on a single-cell basis. With the currently available assays, LSC has proven its wide spectrum of clinical applicability in slide-based cytometry and can be introduced as a standard technology in multiple clinical settings.     

Analysis of apoptosis by laser scanning cytometry

Flow cytometry techniques that are widely used in studies of cell death, and particularly in the identification of apoptotic cells, generally rely on the measurement of a single characteristic biochemical or molecular attribute. These methods fail to recognize cell death lacking that attribute, as in some examples of atypical apoptosis. Since apoptosis was originally defined by morphologic criteria, we suggest that for any new cell system the cytometry-defined apoptosis be confirmed by morphologic examination. This quality assurance measure is now provided by laser scanning cytometry (LSC). LSC measurements of cell fluorescence are precise and highly sensitive, comparable to flow cytometry (FCM), and can be carried out on cells on slides, permitting cell by cell correlation of fluorescence cytometry with visual microscopic morphology. In this report we describe adaptations of various flow cytometry techniques for detection of apoptosis by laser scanning cytometry. We also describe features unique to LSC that are useful in recognizing apoptosis. Hyperchromicity of DNA, reflecting chromatin condensation, is evidenced by high maximal pixel values for fluorescence of the DNA-bound fluorochrome. Mitochondrial probes that have been adapted to LSC to measure the drop in mitochondrial transmembrane potential that occurs early in apoptosis include rhodamine 123, 3,3'-dihexiloxadicarbocyanine [DiOC6(3)], and the aggregate dye 5,5',6,6'tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). The changes in plasma membrane phospholipids and transport function, also early in apoptosis, are probed by a combination of the fluoresceinated annexin V and DNA fluorochromes such as propidium or 7-aminoactinomycin D. We also review methods of detection of apoptosis based on analysis of DNA fragmentation and their application to clinical oncology. Visual examination of the presumed apoptotic cells detected by cytometry makes it possible to discriminate those that are genuine from monocytes/macrophages that have ingested nuclear fragments via apoptotic bodies. Applications of flow cytometry and laser scanning cytometry in analysis of cell death are discussed and their respective advantages and disadvantages compared.     

Mechanism for transition from initial to stable cell-cell adhesion: kinetic analysis of E-cadherin-mediated adhesion using a quantitative adhesion assay

A centrifugal force-based adhesion assay has been used to quantitatively examine the kinetics of formation of cell-cell contacts mediated specifically by expression of E-cadherin under the control of a glucocorticoid-inducible promoter in mouse fibroblasts. Analysis of cells expressing maximal or minimal levels of E-cadherin showed that the strength of E-cadherin-mediated adhesion developed in a single exponential step over a short time (half-maximal adhesion, 13-17 min). At 37 degrees C, adhesion strength increased rapidly in the first 20 min without an apparent lag phase. After 90 min, adhesion strength reached a plateau. Differences in final strengths of adhesion were commensurate with the level of E-cadherin expression. Strengthening of adhesion was temperature dependent. At 19 degrees C, strengthening of adhesion was delayed and subsequently developed with a slower rate compared to adhesion at 37 degrees C. At 4 degrees C, adhesion was completely inhibited. Strengthening of adhesion was absolutely dependent on a functional actin cytoskeleton since adhesion did not develop when cells were treated with cytochalasin D. Together, our current and previous (McNeill et al., 1993.J. Cell Biol. 120:1217-1226) studies indicate that the rate of initial strengthening of E-cadherin- mediated adhesion is neither dependent on the amount of E-cadherin expressed nor on long-range protein diffusion in the membrane to the adhesion site. However, initial strengthening of adhesion is dependent on temperature-sensitive cellular activities that may locally couple clusters of E-cadherin to the actin cytoskeleton.     

A practical method to determine the amount of tissue to analyze using laser scanning cytometry.

BACKGROUND: Laser scanning cytometry (LSC) is a new technology similar to flow cytometry but generates data from analysis of successive microscopic fields. Unlike its use in other applications, LSC-generated data are not random when used for tissue sections, but are dependent on the microanatomy of the tissue and the distribution and expression of the protein under investigation. For valid LSC analysis, the data generated requires the evaluation of a sufficient tissue area to ensure an accurate representation of expression within the tissue of interest. METHODS: In this report, we describe a simple and common sense method for determining the area of tissue required for sound LSC analysis by tracking the variation in the measure of target expression with increasing number of fields until it approaches zero. RESULTS: This approach was used to evaluate the expression of immunohistochemical markers with differing tissue distributions in liver (PMP70, CYP1A2, and Ki67 positive macrophages) and a colorectal adenocarcinoma (activated caspase-3 positive cells), which exhibited diffuse, regional (centrilobular), random, and irregular distribution patterns respectively. CONCLUSIONS: Analyses of these markers demonstrated that the amount of tissue area required to reach a steady measure of a parameter increased with increasing variability of the tissue distribution.     

Measurement of markers for breast cancer in a model system using laser scanning cytometry

BACKGROUND: A variety of markers, including Ki67, estrogen receptors (ER), and progesterone receptors (PgR), are frequently measured in fine needle aspirates (FNA) from human breast carcinomas. We used a human breast carcinoma cell line, MCF7, as a model system to investigate the use of laser scanning cytometry (LSC) for the measurement of these markers. Additionally, we measured the number of apoptotic cells. METHODS: Cells were treated with drugs to vary the expression of markers and the number of apoptotic cells. They were then fixed on microscope slides. For LSC, the cells were stained for the different markers with fluorescein using immunofluorescence and for apoptotic cells using the TUNEL assay. The nuclei were counterstained with propidium iodide. A parallel set of slides was stained using horseradish peroxidase and diaminobenzidine and scored manually by conventional light microscopy. RESULTS: The results from the LSC closely paralleled those obtained by manual scoring of immunohistochemical stains. CONCLUSIONS: It should be possible to use LSC for the routine measurement of nuclear markers in FNAs from human breast carcinomas.     

Proliferation and apoptosis in solid tumors. Analysis by laser scanning cytometry

OBJECTIVE: To examine the relationship between apoptosis and proliferation in a series of human solid malignant tumors, making use of objective, reproducible techniques newly developed for laser scanning cytometry (LSC). STUDY DESIGN: Apoptosis was detected by in situ end labeling of DNA strand breaks with FITC-conjugated nucleotide. Proliferation was detected by Ki-67 antibody. Two parameters were detected independently and simultaneously with DNA measurement on aliquots of cell suspensions obtained by mechanical dissociation of fresh tumors and placed on microscope slides. RESULTS: The number of cells undergoing apoptosis varied from 0.5% to 28.1% (average, 5.4 +/- 6.0). Aneuploid tumors showed a higher percentage of apoptotic cells (7.9 +/- 7.2) as compared to diploid tumors (3.4 +/- 4.0). Tumors with the greatest number of apoptotic cells on LSC also had the largest number of apoptotic cells on light microscopic examination. The number of cells labeled by Ki-67 ranged from 1.7% to 56.7% (average, 20.0 +/- 15.5). Aneuploid tumors were characterized by a higher Ki-67 index (average, 28.3 +/- 14.3%) than the diploid tumors (13.2 +/- 13.3%). CONCLUSION: Overall, there was a very weak or no correlation between apoptosis and proliferation. However, a subset of aneuploid tumors had a high percentage of cells positive for Ki-67 and low percentage of apoptotic cells. Diploid tumors did not show any correlation between apoptosis and proliferation, although many of those tumors had both low apoptotic and proliferative indices. Whether those differences are of prognostic significance remains to be determined in follow-up studies that include more cases and clinical data. Here we have shown that LSC is a powerful new tool of potential clinical value for fast, objective analysis of apoptosis, proliferation and DNA ploidy in solid malignant tumors.     

Measurement of DNA damage associated with apoptosis by laser scanning cytometry.

BACKGROUND: Phosphatidylserine (PS) binding by annexin V (AV) is an early membrane marker of apoptosis. Using laser scanning cytometry (LSC) and the comet assay, we showed that the DNA of AV(+) cells is so highly fragmented that it cannot be quantified by the comet assay (Bacso et al.: Cancer Res 60:4623-8, 2000). METHODS: The "halo" assay was used instead of the comet assay to quantify DNA damage associated with apoptosis. The LSC was used to measure both AV fluorescence and DNA damage on the same Jurkat cells following treatment with anti-Fas. The data from both sets of measurements were merged, allowing direct correlation of membrane and nuclear markers of cell death. RESULTS: AV(+) cells had significant DNA damage determined by the ratio between nuclear DNA and peripheral (migrated) DNA. Cells in the early and late stages of apoptosis could be discriminated on the basis of DNA content. In addition, it was possible to distinguish between apoptotic and necrotic cells in the AV(+) propidium iodide-positive population based on DNA content and DNA damage. The addition of specific inhibitors for caspases-8, 9, and 3 blocked both PS externalization and DNA fragmentation, indicating these events are downstream from caspase activation. CONCLUSIONS: This technique allows accurate distinction between apoptotic and necrotic cells and cytometric grading of apoptosis.     

Inference of cell cycle-dependent proteolysis by laser scanning cytometry

Mechanisms that couple protein turnover to cell cycle progression are critical for coordinating the events of cell duplication and division. Despite the importance of cell cycle-regulated proteolysis, however, technologies to measure this phenomenon are limited, and typically involve monitoring cells that are released back into the cell cycle after synchronization. We describe here the use of laser scanning cytometry (LSC), a technical merger between fluorescence microscopy and flow cytometry, to determine cell cycle-dependent changes in protein stability in unperturbed, asynchronous, cultures of mammalian cells. In this method, the ability of the LSC to accurately measure whole cell fluorescence is employed, together with RNA fluorescence in situ hybridization and immunofluorescence, to relate abundance of a particular RNA and protein in a cell to its point at the cell cycle. Parallel monitoring of RNA and protein levels is used, together with protein synthesis inhibitors, to reveal cell cycle-specific changes in protein turnover. We demonstrate the viability of this method by analyzing the proteolysis of two prominent human oncoproteins, Myc and Cyclin E, and argue that this LSC-based approach offers several practical advantages over traditional cell synchronization methods.     

Use of laser scanning cytometry to study tumor microenvironment

The study of phenomena occurring in the tumor microenvironment is a challenging task because of technical difficulties, particularly when dealing with hypocellular specimens. Laser scanning cytometry (LSC) is a new laboratory technology that has been recently introduced to overcome the limitations of other traditional technologies. By combining the properties and the advantages of flow cytometry (FC) and immunohistochemistry (IHC), LSC allows the investigator to obtain objective information on DNA content, protein expression and cellular localization is combination with morphological features. It has been already shown that LSC results are reliable compared to more traditional technologies, and its implementation in the clinical routine is under way. Its use in oncology, which is rapidly expanding, spans from apoptosis analysis to DNA content quantitation and tumor cell phenotyping. Here we describe the technology underlying this novel fluorescence-based device, review its use in oncology by dissecting the phenomena occurring in the tumor microenvironment and propose its application for the immunological follow-up of malignant lesions undergoing immunotherapeutic manipulation.     

Mechanism of cell-cell adhesion complex assembly.

Cell-cell adhesion complexes play an important role in the organization and behavior of cells in tissues. An important step in the formation of such complexes is the clustering of the adhesion receptors; this is critical for proper adhesion, for anchorage of the cytoskeleton to the plasma membrane, and for generation of different intracellular signals. Recent advances reveal that several interconnected mechanisms are responsible for clustering of the different adhesion receptors.     

Unique analytical capabilities of laser scanning cytometry (LSC) that complement flow cytometry

Flow cytometry has become an indispensable instrumentation in many disciplines of biology and medicine. There are some limitations of flow cytometry, inherent to the fact that the cells are measured in flow, which limit its usefulness in some applications. The microscope-based laser scanning cytometer (LSC) has many features similar to flow cytometry but few restrictions of the latter and therefore it is useful in many new applications. This review briefly outlines the applications that are unique to LSC, particularly related to its morphometric capabilities and the possibility of cell relocation. Potential future applications of LSC are also discussed.     

Three-dimensional DNA image cytometry by confocal scanning laser microscopy in thick tissue blocks.

A method for the quantification of nuclear DNA in thick tissue blocks by confocal scanning laser microscopy is presented. Tissues were stained en bloc for DNA by chromomycin A3. Three-dimensional images, 60 microns deep, were obtained by stacking up confocal fluorescent images obtained with an MRC-500 (Bio-Rad, Richmond, CA). The effects due to bleaching and attenuation by depth of fluorescence emission were corrected mathematically. The DNA contents were estimated by summing up the detected emission intensities (discretized into pixel gray levels) from each segmented nucleus. Applications to an adult rat liver and to a human in situ carcinoma of theesophagus are shown to demonstrate, respectively, the precision of the method and its potential usefulness in histopathology. Comparisons are made with DNA histograms obtained on the same materials by image cytometry on smears and by flow cytometry. Ploidy peaks obtained with the confocal method, although wider than with other methods, are well separated. Confocal image cytometry offers the invaluable advantage of preserving the tissue architecture and therefore allowing, for instance, the selection of histological regions and the evaluation of the degree of heterogeneity of a tumor.     

Development of a stereological method to measure levels of fluoropyrimidine metabolizing enzymes in tumor sections using laser scanning cytometry.

BACKGROUND: The enzymes thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) influence the activities of fluoropyrimidine anticancer drugs. The sensitivity of cancer cells to capecitabine, which is an oral, tumor-selective pre-prodrug of 5-fluorouracil may correlate better to the TP/DPD ratio than to levels of either enzyme alone. Our goal was to develop a quantitative immunofluorescent method for estimating the levels of TP, DPD, and their ratio in archival tumor sections. METHODS: Mouse anti-TP and rat anti-DPD monoclonal antibodies were used for parallel indirect immunofluorescent staining. The fluorescence was measured using a laser scanning cytometer (LSC; CompuCyte, Cambridge, MA) in single cells and in sections prepared from cell lines and a human tumor. The phantom contouring feature of the LSC provided a stereologic approach for collecting the fluorescence intensity data from sections. RESULTS: The relative fluorescence intensities measured in single cells or in sections of the cell lines, using single or double labeling, were similar, supporting the suitability of phantom contouring and two-color staining. Sections of the T-24 and ZR-75-1 cell lines placed on the same slide as the tumor section were used as internal standards for fluorescence measurements. The TP/DPD ratios measured in three cell lines correlated well with the cytotoxicity of 5'-deoxy-5-fluorouridine measured in vitro, indicating that the measurements are related to the biological activity of the drug. CONCLUSIONS: Plotting the data as contour maps of the topologic distribution of fluorescence intensities in tumor sections allows subsequent histopathologic examination, which may reveal features of the tumors leading to high or low ratios of these enzymes. In addition, this method can be used for any drug target/metabolic system where the key components are known and suitable antibodies are available.     

Multi-photon laser scanning microscopy using an acoustic optical deflector

Multi-photon laser scanning microscopes have many advantages over single-photon systems. However, the speed and flexibility of currently available multi-photon microscopes are limited by the use of mechanical mirrors to steer pulsed radiation for fluorophore excitation. Here, we describe the multi-photon adaptation of a confocal microscope that uses an acoustic optical deflector (AOD) for beam steering. AODs are capable of very rapid scanning and, in addition, offer the flexibility of zooming, panning, and being adjustable for slow image acquisition. Because of the highly dispersive nature of AODs, pulsed radiation must be temporally compressed by introducing negative dispersion into the beam path. More critically, pulsed radiation must also be spatially compressed by introducing lateral dispersion into the beam path. This was accomplished by using two prisms in the external beam path and by introducing a third prism element subsequent to the AOD. The end result is an AOD-based multi-photon microscope that is capable of rapid imaging of physiological events as well as slow detection of weakly fluorescent biological samples.     

Violet laser diodes in flow cytometry: an update.

INTRODUCTION: In previous studies we and others have demonstrated the usefulness of violet laser diodes (VLDs) as replacement laser sources for krypton-ion lasers on stream-in-air cytometers. Previously available VLDs had a maximum available power of less than 25 mW; this was sufficient for excitation of densely labeled cell surface antigens using fluorochromes such as Cascade Blue or Pacific Blue, but may have been insufficient for applications requiring higher levels of photon saturation, such as low-level expression of Cyan Fluorescent Protein (ECFP) in CFP-YFP FRET applications. In this follow-up study, we have tested more powerful VLDs emitting at 55 mW, and a beam-merged dual module VLD with 100 mW combined output, for their ability to excite a variety of violet-excited fluorochromes, including CFP. METHODS: A dual module VLD (two linear polarized VLDs with their beams merged by a polarized beam combiner) emitting at 404 nm was mounted on a BD FACSVantage DiVa stream-in-air cytometer. The individual polarized 55 mW beams or the 100 mW combined beams were used to analyze PBMCs labeled with the violet-excited probes Cascade Blue, Alexa Fluor 405, Cascade Yellow and Pacific Orange dyes. Violet-excited fluorescent microsphere mixtures with decreasing fluorescence levels were also used to detect the minimum sensitivity threshold and precision of these lasers. VLD excitation on a gel-coupled cuvette flow cytometer was used as a sensitivity baseline. RESULTS: The dual module 100 mW VLD gave both sensitivity and precision levels approaching that observed for lower-power sources on a cuvette cytometer. Single polarized VLD modules at 55 mW gave slightly decreased sensitivity for the microspheres standards and all the tested fluorochromes compared to the 100 mW source. CONCLUSIONS: While 55 mW laser sources performed adequately in the stream-in-air format, increasing the power to 100 mW did give a small but detectable increase in instrument sensitivity. This sensitivity level approached that of cuvette systems.     

Quantification of T-cell-mediated apoptosis in heterogeneous leukemia populations using four-color multiparameter flow cytometry.

BACKGROUND: The unique capacity of dendritic cells to present antigens to naive T cells is being increasingly utilized in cancer therapy. The efficacy of cell-based immunotherapy can be analyzed by determination of cytotoxic activity of T cells toward tumor cells in vitro. This study supplies a flow cytometric method to analyze T-cell-mediated cytotoxic activity toward heterogeneous leukemic cell populations at a single-cell level. METHODS: The fluorescent probe SYTO16 and the dead-cell dye 7-aminoactinomycine-D (7-AAD) were used to identify early and late stages of apoptosis in combination with leukemia cell-identifying markers. Determination of viable, apoptotic, and necrotic cells was performed by inclusion of fluorescent beads. RESULTS: In nine acute myeloid leukemia samples and three leukemic cell lines the use of SYTO16 next to the dead-cell marker 7-AAD significantly increased (P = 0.001) the sensitivity of the cytotoxicity assay as compared with single use of 7-AAD. Analysis of several effector-to-target ratios revealed the ability to determine dose-response effects. Enumeration of absolute numbers resulted in coefficients of variation of 4.1% and 8.4% for cell lines and leukemic samples, respectively. CONCLUSION: The presented flow cytometric cytotoxicity assay enables the study of T-cell-mediated apoptosis in a heterogenous leukemia population.     

Changes in myoblast membrane electrical properties during cell-cell adhesion and fusion in vitro

Characteristic of the process of myogenesis are the changes in the composition and organization of the cell membrane. While poorly understood, these changes have biochemical and biophysical relevance. Recently, changes in molecular order of the myoblast membrane which accompany differentiation in vitro have been observed (Santini, M.T., Indovina, P.L. and Hausman, R.E. (1987) Biochim. Biophys. Acta 896, 19–25). To further investigate these cell fusion processes we have examined additional physical parameters: conductivity and permittivity of the myoblast membrane during differentiation which reflect the molecular arrangement of the membrane. The determination of these parameters is possible because in the radio frequency range suspensions of cells in an electrolyte buffer show a characteristic conductivity dispersion due to the interfacial polarization. An analysis of our experimental data based on a ‘single-shell’ model showed that conductivity and permittivity of the membrane of pre- and post-fusion myoblasts varied significantly and abruptly. The conductivity of the cell interior (cytosol) remained constant. We discuss the significance of the observed changes in these membrane parameters for myogenesis.