Effects of different detachment procedures on viability,nitroxide reduction kinetics and plasma membrane heterogeneity of V‐79 cells

Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V‐79 cells in the plateau phase of growth (arrested in G₁). We have measured cell viability by a dye‐exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin‐probe MeFASL(10,3) (5‐doxylpalmitoyl‐methylester), which partitions mainly in cell membranes and the hydrophilic spin‐probe TEMPONE (4‐oxo‐2,2,6,6‐tetramethylpiperidine‐1‐oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin‐probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin‐treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V‐79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged.     

The effect of culture conditions on the fluidity of mouse neuroblastoma membranes as estimated by spin label studies

Gas chromatography and spin labeling were used to estimate the composition and fluidity of the lipid acyl chains in intact cell membranes of mouse C1300 neuroblastoma. Neurite formation and an increase in the specific activity of acetylcholinesterase are induced in this cell line by the removal of serum from the culture medium for one day prior to harvesting the cells. The fatty acid composition of induced cells was not significantly different from that of the non-induced controls. Each of the three fatty acid spin labels used in this study indicate that the membranes of induced cells are slightly more fluid than the membranes of control cells. This difference in fluidity appears to be a result of a reduction in the progressive decrease in fluidity occurring during growth in serum. A four-fold reduction in cell viability had no effect on the measured bilayer fluidity. Thus the changes in fluidity appear to be due to the presence or absence of serum rather than to cell type, age, or viability.     

The cytotoxic and cytolytic activity of equinatoxin II from the sea anemone Actinia equina

The cytotoxic and cytolytic effects of equinatoxin II (EqT II) from the sea anemone Actinia equina L. were studied on exponentially growing and synchronized V-79-379 A cell line in culture. The cell viability test and the determination of the cytolytic effect by cell counting confirmed both cytotoxic and cytolytic activity of EqT II. Additionally, cytocidal and cytostatic effects depending on the toxin concentration were observed. The presence of fetal calf serum in the cell culture medium reduced both cytocidal and cytostatic effects by two magnitudes and prevented cytolysis. Combining EqT II and serum resulted in an insoluble complex which was cytostatic even when isolated and resuspended in the culture medium, while the supernatant retained both cytocidal and cytostatic activity. No significant difference in sensitivity between synchronized and exponentially growing cells could be detected after EqT II treatment.     

Modulation of membrane receptor endocytosis by chemical effectors of membrane fluidity

Several chemical effectors were used to induce changes in spleen B cell membrane fluidity. Membrane fluidity was monitored by fluorescence polarization analysis of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH) and cell viability was checked not to be affected by the treatments. Membrane immunoglobulin (Ig) endocytosis by the living B cells with modified or unmodified membranes was quantitatively measured by flow cytometry, using a previously described method (Métézeau et al., 1982, 1984). The kinetics of endocytosis of membrane Ig was not affected by chemical effectors increasing membrane fluidity. On the contrary, increasing membrane microviscosity resulted in the slowing down and eventually the blocking of membrane Ig endocytosis. It is suggested that a step depending on membrane microviscosity is involved in the process of endocytosis; this step may become rate limiting when membranes are artificially rendered or naturally become (i.e. for pathological or particularly differentiated cells) more viscous.     

Morphological changes of V-79 cells after equinatoxin II treatment.

Morphological observations on the V-79-379 A cells after treatment with equinatoxin II (EqT II), isolated from the sea anemone Actina equina L., and fetal calf serum (FCS) treated toxin were examined by transmission electron microscopy. Our results showed that the cells incubated with FCS treated EqT II were almost ultrastructurally unaltered. When the cells were treated with low concentrations of EqT II alone cell ultrastructure was altered with the evidence of numerous blebs and decreased microvilli number on the cell surface and appearance of numerous vesicles in the Golgi regions. High concentrations of EqT II caused disintegration of plasmalemma and intracellular membranes as well as degradation of cytosol.     

14C-Methylated beta-casein as a substrate for plasmin, and its application to the study of milk protein transformations

Electron spin resonance (ESR) spin-label methods were used with 5-doxyl-stearic acid as a probe to investigate membrane fluidity of Chinese hamster ovary (CHO) cells during the cell cycle. A 35 GHz ESR technique was developed to study membrane fluidity of intact cells. This technique requires only about 1/6 the amount of cells compared to the conventional spin-label techniques. With this technique we observed a cyclic change of membrane fluidity during the cell cycle of CHO cells: cells in mitosis had the highest membrane fluidity, whereas cells in G1 and early S phases had the lowest membrane fluidity.     

ESR studies on membrane fluidity of Chinese hamster ovary cells grown on microcarriers and in suspension

Electron spin resonance (ESR) spin label methods were used to study membrane fluidity of Chinese hamster ovary (CHO) cells grown on microcarriers and in suspension using 5-doxylstearic acid spin label as a probe. CHO cells grown on microcarriers had a more rigid cell membrane compared to CHO cells grown in suspension culture. CHO cells removed from the surface of the microcarriers by either trypsinization, EDTA treatment or osmotic shock had a membrane fluidity similar to that of CHO cells grown in suspension culture. Conversely, when the cells grown in suspension culture were attached and flattened on the surface of the microcarriers the fluidity decreased. Moreover, membrane fluidity of CHO cells grown on microcarriers changed as a function of the population density, whereas that of the cells in suspension did not. This implies that cell adhesion and/or cell-cell interactions influence the fluidity of the cell surface membrane.     

Electron spin resonance studies of changes in membrane fluidity of Chinese hamster ovary cells during the cell cycle

Electron spin resonance (ESR) spin-label methods were used with 5-doxyl-stearic acid as a probe to investigate membrane fluidity of Chinese hamster ovary (CHO) cells during the cell cycle. A 35 GHz ESR technique was developed to study membrane fluidity of intact cells. This technique requires only about $\text{1}\text{6}$ the amount of cells compared to the conventional spin-label techniques. With this technique we observed a cyclic change of membrane fluidity during the cell cycle of CHO cells: cells in mitosis had the highest membrane fluidity, whereas cells in G₁ and early S phases had the lowest membrane fluidity.     

Fluorescence-based cell viability screening assays using water-soluble oxygen probes

A simple luminescence-based assay for screening the viability of mammalian cells is described, based on the monitoring of cell respiration by means of a phosphorescent water-soluble oxygen probe that responds to changes in the concentration of dissolved oxygen by changing its emission intensity and lifetime. The probe was added at low concentrations (0.3 microM to 0.5 nM) to each sample containing a culture of cells in the wells of a standard 96-well plate. Analysis of oxygen consumption was initiated by applying a layer of mineral oil on top of each sample followed by monitoring of the phosphorescent signal on a prompt or time-resolved fluorescence plate reader. Rates of oxygen uptake could be determined on the basis of kinetic changes of the phosphorescence (initial slopes) and correlated with cell numbers (10(5) to 10(7) cells/mL for FL5.12 lymphoblastic cell line), cell viability, or drug/effector action using appropriate control samples. The assay is cell noninvasive, more simple, robust, and cost-effective than existing microplate-based cell viability assays; is compatible with existing instrumentation; and allows for high-throughput analysis of cell viability.     

Effects of divalent cations and polyethylene glycol on the membrane fluidity of protoplast

Calcium is often used to stabilize membranes and enhance membrane fusion. We have used the fatty acid spin label, 5-nitroxy stearic acid to measure fluidity changes in the plasma membrane of carrot suspension culture cell protoplasts in response to divalent cations. Electron spin resonance spectra from spin-labeled protoplasts showed no membrane fluidity changes (as determined by the hyperfine splitting constant, 2A_max) in the presence of Mg from 0 to 10 millimolar or Ca from 0 to 5 millimolar. Protoplasts in 10 millimolar Ca, however, showed a dramatic increase of 5 gauss in 2A_max and evidence of exchange-broadening. The original (control) spectrum was regained by removing bound Ca with a Ca chelator. Polyethylene glycol, which enhances protoplast fusion, did not alter the membrane fluidity in the region of the 5-nitroxy stearic acid probe if added simultaneously with or following 10 millimolar Ca. Pretreatment with polyethylene glycol did, however, inhibit the Ca-induced phase separation. These data on a living system describe membrane structural changes under conditions similar to those used for protoplast fusion.     

Rates of chilling to 0°C: implications for the survival of microorganisms and relationship with membrane fluidity modifications

The effects of slow chilling (2°C min⁻¹) and rapid chilling (2,000°C min⁻¹) were investigated on the survival and membrane fluidity of Escherichia coli, of Bacillus subtilis, and of Saccharomyces cerevisiae. Cell death was found to be dependent on the physiological state of cell cultures and on the rate of temperature downshift. Slow temperature decrease allowed cell stabilization, whereas the rapid chilling induced an immediate loss of viability of up to more than 90 and 70% for the exponentially growing cells of E. coli and B. subtilis, respectively. To relate the results of viability with changes in membrane physical state, membrane anisotropy variation was monitored during thermal stress using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene. No variation in the membrane fluidity of all the three microorganisms was found after the slow chilling. It is interesting to note that fluorescence measurements showed an irreversible rigidification of the membrane of exponentially growing cells of E. coli and B. subtilis after the instantaneous cold shock, which was not observed with S. cerevisiae. This irreversible effect of the rapid cold shock on the membrane correlated well with high rates of cell inactivation. Thus, membrane alteration seems to be the principal cause of the cold shock injury.     

Combined cold, acid, ethanol shocks in Oenococcus oeni: Effects on membrane fluidity and cell viability

The effects of combined cold, acid and ethanol on the membrane physical state and on the survival of Oenococcus oeni were investigated. Membrane fluidity was monitored on intact whole O. oeni cells subjected to single and combined cold, acid and ethanol shocks by using fluorescence anisotropy with 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe. Results showed that cold shocks (14 and 8 °C) strongly rigidified plasma membrane but did not affect cell survival. In contrast, ethanol shocks (10-14% v/v) induced instantaneous membrane fluidisation followed by rigidification and resulted in low viability. Acid shocks (pH 4.0 and pH 3.0) exerted a rigidifying effect on membrane without affecting cell viability. Whatever the shock orders, combined cold (14 °C) and ethanol (14% v/v) shocks resulted in strong membrane rigidification. Interestingly, O. oeni survived combined cold and ethanol shocks more efficiently than single ethanol shock. Membrane rigidification was induced by ethanol-and-acid (10% v/v - pH 3.5) shock and correlated with total cell death. In contrast, O. oeni recovered its viability when subjected to cold (8 °C)-then-ethanol-and-acid shock which strongly rigidified the membrane. Our results suggested a positive short-term effect of combined cold, acid and ethanol shocks on membrane fluidity and viability of O. oeni.     

Combined cold, acid, ethanol shocks in Oenococcus oeni: effects on membrane fluidity and cell viability

The effects of combined cold, acid and ethanol on the membrane physical state and on the survival of Oenococcus oeni were investigated. Membrane fluidity was monitored on intact whole O. oeni cells subjected to single and combined cold, acid and ethanol shocks by using fluorescence anisotropy with 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe. Results showed that cold shocks (14 and 8 degrees C) strongly rigidified plasma membrane but did not affect cell survival. In contrast, ethanol shocks (10-14% v/v) induced instantaneous membrane fluidisation followed by rigidification and resulted in low viability. Acid shocks (pH 4.0 and pH 3.0) exerted a rigidifying effect on membrane without affecting cell viability. Whatever the shock orders, combined cold (14 degrees C) and ethanol (14% v/v) shocks resulted in strong membrane rigidification. Interestingly, O. oeni survived combined cold and ethanol shocks more efficiently than single ethanol shock. Membrane rigidification was induced by ethanol-and-acid (10% v/v - pH 3.5) shock and correlated with total cell death. In contrast, O. oeni recovered its viability when subjected to cold (8 degrees C)-then-ethanol-and-acid shock which strongly rigidified the membrane. Our results suggested a positive short-term effect of combined cold, acid and ethanol shocks on membrane fluidity and viability of O. oeni.     

EPR spin labeling measurements of thylakoid membrane fluidity during barley leaf senescence

Physical properties of thylakoid membranes isolated from barley were investigated by the electron paramagnetic resonance (EPR) spin labeling technique. EPR spectra of stearic acid spin labels 5-SASL and 16-SASL were measured as a function of temperature in secondary barley leaves during natural and dark-induced senescence. Oxygen transport parameter was determined from the power saturation curves of the spin labels obtained in the presence and absence of molecular oxygen at 25 °C. Parameters of EPR spectra of both spin labels showed an increase in the thylakoid membrane fluidity during senescence, in the headgroup area of the membrane, as well as in its interior. The oxygen transport parameter also increased with age of barley, indicating easier diffusion of oxygen within the membrane and its higher fluidity. The data are consistent with age-related changes of the spin label parameters obtained directly by EPR spectroscopy. Similar outcome was also observed when senescence was induced in mature secondary barley leaves by dark incubation. Such leaves showed higher membrane fluidity in comparison with leaves of the same age, grown under light conditions. Changes in the membrane fluidity of barley secondary leaves were compared with changes in the levels of carotenoids (car) and proteins, which are known to modify membrane fluidity. Determination of total car and proteins showed linear decrease in their level with senescence. The results indicate that thylakoid membrane fluidity of barley leaves increases with senescence; the changes are accompanied with a decrease in the content of car and proteins, which could be a contributing factor.     

Membrane lipid dynamics and density dependent growth control in normal and transformed avian cells

Membrane fluidity of normal chick embryo fibroblasts and normal Japanese quail fibroblasts and their Rous sarcoma virus and methylcholanthrene transformed counterparts was investigated using the technique of fluorescence depolarisation of 1,6-diphenylhexatriene incorporated in the whole cells and in their isolated plasma membrane vesicles. Normal cells and isolated plasma membranes of normal cells showed significant changes in fluidity as a function of population density while neither Rous sarcoma virus transformed nor methylcholanthrene tumor cells or their isolated plasma membrane showed this effect. Stimulation of growth by addition of calf serum to cultures of quiescent, density-inhibited normal cells was accompanied by rapid changes in the direction of increased membrane lipid fluidity. Neither sparse normal cells, nor sparse or dense transformed cells showed any significant fluidity change in their membrane lipids upon addition of serum. Enzyme and electron microscopic analysis of the ratios of different membrane types in each cell type showed that this ratio was invariant with respect to cell population density but different between transformed and normal cells. Hence, the fluidity changes observed, measured as the mean rotational correlation time of the fluorescene probe in the membrane lipids, truly reflect organisational differences, occurring as a function of population density in cultures of cells which retain density-dependent growth control.     

Spin label studies on rat liver and heart plasma membranes: Effects of temperature,calcium, and lanthanum on membrane fluidity

The structures of rat liver and heart plasma membranes were studied with the 5-nitroxide stearic acid spin probe, I(1 2,3). The polarity-corrected order parameters (S) of liver and heart plasma membranes were independent of probe concentration only if experimentally determined low I(1 2,3)/lipid ratios were employed. At higher probe/lipid ratios, the order parameters of both membrane systems decreased with increasing probe concentration, and these effects were attributed to enhanced nitroxide radical interactions. Examination of the temperature dependence of approximate and polarity-corrected order parameters indicated that lipid phase separations occur in liver (between 19° and 28°C) and heart (between 21° and 32°C) plasma membranes. The possibility that a wide variety of membrane-associated functions may be influenced by these thermotropic phase separations is considered. Addition of 3.9 mM CaCl₂ to I(1 2,3)-labeled liver plasma membrane decreased the fluidity as indicated by a 5% increase in S at 37°C. Similarly, titrating I(1 2,3)-labeled heart plasma membranes with either CaCl₂ or LaCl₃ decreased the lipid fluidity at 37°C, although the magnitude of the La³⁺ effect was larger and occurred at lower concentrations than that induced by Ca²⁺; addition of 0.2 mM La³⁺ or 3.2 mM Ca²⁺ increased S by approximately 7% and 5%, respectively. The above cation effects reflected only alterations in the membrane fluidity and were not due to changes in probe–probe interactions. Ca²⁺ and La³⁺ at these concentrations decrease the activities of such plasma membrane enzymes as Na⁺, K⁺-ATPase and adenylyl cyclase, and it is suggested that the inhibition of these enzymes may be due in part to cation-mediated decreases in the lipid fluidity.     

The decreased membrane fluidity of in vivo aged,human erythrocytes. A spin label study

The decreased membrane fluidity of the in vivo aged, human erythrocytes is found, by monitoring the electron paramagnetic resonance (EPR) spectra of fatty acid spin labels incorporated into the membrane.In addition, the decreased cell sizes and the decreased cholesterol and phospholipids contents, without significant changes of the quantity of the membrane proteins, also the decrease of ATP and 2,3-diphosphoglycerate and the increase of ADP and AMP, in the aged cells, were observed. Further the functional impairments of the aged cells, i.e. the increased oxygen affinity and the decreased deformability, were shown.On the basis of these quantitative data, the alteration of the protein-lipid organization, due to decreased lipid/protein ratio, the modified protein-lipid interaction and/or the influences of the diminished ATP content, is suggested to contribute towards the decreased membrane fluidity of the in vivo aged erythrocytes.     

The decreased membrane fluidity of in vivo aged, human erythrocytes. A spin label study.

The decreased membrane fluidity of the in vivo aged, human erythrocytes is found, by monitoring the electron paramagnetic resonance (EPR) spectra of fatty acid spin labels incorporated into the membrane. In addition, the decreased cell sizes and the decreased cholesterol and phospholipids contents, without significant changes of the quantity of the membrane proteins, also the decrease of ATP and 2,3-diphosphoglycerate and the increase of ADP and AMP, in the aged cells, were observed. Further the functional impairments of the aged cells, i.e. the increased oxygen affinity and the decreased deformability, were shown. On the basis of these quantitative data, the alteration of the protein-lipid organization, due to decreased lipid/protein ratio, the modified protein-lipid interaction and/or the influences of the diminished ATP content, is suggested to contribute towards the decreased membrane fluidity of the in vivo aged erythrocytes.     

The oxidizing agent menadione induces an increase in the intracellular molecular oxygen concentration in K562 and A431 cells: Direct measurement using the new paramagnetic EPR probe fusinite

The intracellular molecular oxygen concentration in control and menadione-treated K562 (an erythroleukemic cell line that grows in suspension) and A431 (an epidermal carcinoma that grows in monolayer) cells was measured directly by using the new electron paramagnetic resonance (EPR) probe fusinite. Because the oxidizing agent menadione is known to damage mitochondria and the cytoplasmic membrane in other cell systems, before conducting measurements of oxygen concentration in K562 and A431 cells, it was necessary to establish injury in these systems as well. Consequently, morphological and flow cytometric analyses were conducted after menadione treatment. The data presented here show that the two cell lines are heavily damaged by menadione. Once this menadione-induced injury was demonstrated, measurements of oxygen concentration were carried out in both K562 and A431 cells. Treatment with this quinone induces a sharp increase in intracytoplasmic molecular oxygen in both cell lines (from about 1% to about 10 and 15% in K562 and A431 cells, respectively). In addition, to gain a more complete understanding of the effects of menadione on cells, the extracellular molecular oxygen concentration and the oxygen consumption rate were also measured in control and menadione-treated K562 cells. These measurements demonstrate that menadione treatment results in an increase in the extracellular oxygen concentration (from about 5% in controls to 15% in treated cells) as well as a decrease in the oxygen consumption rate (from about 10 ng O/min/10⁶ cells in controls to 3 ng O/min/10⁶ cells after menadione exposure). The importance of the new EPR probe fusinite in monitoring directly cellular functions in which oxygen is involved and the effects of menadione on cellular oxygen balance are discussed.     

Aging of the erythrocyte IV. Spin-label studies of membrane lipids,proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.