Differences in the surface properties of the mating types of Physarum polycephalum

In the slime mold Physarum polycephalum, formation of a diploid plasmodium occurs when compatible haploid amoebae fuse. To study cell surface changes associated with the fusion process, a non-destructive method known as aqueous, two-phase partitioning was employed. Using a two-phase system of dextran and polyethylene glycol, we observed that the two mating types (RSD4 and MA185) have different partition coefficients and hence different surface properties. Based on their partitioning behavior, MA185 cells appear to have a more hydrophobic surface than RSD4 amoeba. The partition coefficient of both cell types decreased with time. If amoebae were maintained in culture until they encysted, differences in their surface were not detectable.     

Characteristics of hemagglutination reaction in methanotrophic bacteria

The reaction of hemagglutination with trypsin-treated rabbit erythrocytes was used to reveal lectins on the cell surface of methanotrophic bacteria and in their culture liquids. By this method, no lectins were detected on the cell surface of Methylococcus capsulatus IMV B-3001 and Methylomonas rubra IMV B-3075 or in the culture liquid of any of the species studied. With intact cells of Methylocystis parvus IMV B-3491, the positive hemagglutination reaction observed was nonspecific and most probably occurred due to the high cell surface hydrophobicity characteristic of this species.     

A high-throughput hybridoma selection method using fluorometric microvolume assay technology

Monoclonal antibodies (mAb) are not only useful reagents but also represent a promising type of therapeutics due to their high affinity and exquisite specificity for their antigens. A critical step in mAb generation is to identify antigen-specific antibodies. Although enzyme-linked immunosorbent assay (ELISA) has been broadly applied for antibody selection against secreted antigens, an inherent disadvantage for ELISA is the difficulty in identifying antibodies that recognize the native conformation of cell surface antigens. To overcome this drawback, the authors have developed a high-throughput cell-based antibody binding assay using fluorometric microvolume assay technology (FMAT). This method offers a homogeneous assay for detection of antibody binding to its antigen on the cell surface. To distinguish antibodies that bind to antigen on the cell surface from those that bind nonspecifically to cells, the binding is assessed using both antigen-expressing cells and related cells devoid of the antigen expression. This assay can detect antibodies at a concentration as low as 5 ng/mL and cell surface antigen as low as 9000 copies per cell. Results demonstrate that the FMAT method provides a sensitive and homogeneous assay to detect antibody binding to cell surface antigens and is amenable for high-throughput hybridoma selection.     

Rapid method for monitoring maximum biosurfactant production obtained by acetone precipitation

Summary The appearance of surface active compounds in the culture broths grown on hydrocarbons can be easily monitored by measuring the surface tension of the cell free broth. Using three bacterial strains it was found that yields of the biosurfactants were maximum at the time when lowest surface tension values were recorded. Thus reduction of surface tension of the medium is a rapid method for assay of maximum biosurfactant formation prior to their actual isolation.     

An immunogold-silver staining method for detection of cell surface antigens in cell smears

We developed an indirect immunogold-silver staining method for detection of leukocyte cell surface antigens in cell smears. Air-dried and fixed cytocentrifuge preparations or smears of peripheral blood leukocytes were incubated with monoclonal antibodies (MAb) and colloidal gold-labeled secondary antibodies. The preparations were post-fixed and silver enhancement was performed. The smears were counterstained with May-Grunwald-Giemsa and examined in brightfield light microscopy. The morphology of the cells was well preserved. Leukocytes reacting with the MAb showed black granules on their surface membranes. The intense immunostaining and the low background allowed a rapid enumeration of the positive cells. The labeling could be detected with high sensitivity by epipolarization microscopy. This immunogold-silver staining method was used to quantify T- and B-lymphocytes and natural killer cells in buffy coat smears of normal adult blood. These lymphocyte subsets correlated well with those obtained in smears with the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method and with those found by labeling of mononuclear cells in suspension with immunogold-silver staining. This immunogold-silver staining method forms a good alternative to immunoenzyme methods for study of hematologic cells. In addition, it could be a general procedure for detection of cell surface antigens in all kinds of cell smears.     

Comparison of several digital and stereological methods for estimating surface area and volume of cells studied by confocal microscopy.

BACKGROUND: The implementation of different methods for estimating the surface area and volume of cells studied by confocal microscopy was developed. The methods were compared from the point of view of their precision, applicability and efficiency. METHODS: Interactive stereological methods (spatial grid method, fakir method, Cavalieri principle) as well as automatic digital methods (digital Crofton method, voxel counting, triangulation method, iso-intensity contouring method) were considered. The methods were tested on model geometrical solids and on real volume images consisting of a stack of serial sections encompassing entire tobacco BY-2 cells or cell chains. RESULTS: It is shown that many of the studied methods are very precise when applied to cells of simple or moderately complex shapes. The automatic digital methods are fast and precise but their applicability is limited by the necessity to segment automatically the object surface and to find an optimal resolution. This limitation is not present in stereological methods which are applied interactively and thus are more time-consuming. CONCLUSIONS: The presented implementations of the fakir method and the Cavalieri principle enable interactive, unbiased and efficient estimation of the cell surface area and volume. The recommended steps for measuring the surface area and/or volume of objects studied by confocal microscopy are described.     

Lectin-binding spectra in the hyperplastic human tonsil. Effect of formalin fixation and paraffin embedding on lectin affinity of tissue components

In order to determine the effect of routine fixation on the lectin affinity of tissue structures, we used unconjugated lectins and an indirect immunoalkaline-phosphatase method for frozen sections, and the peroxidase-anti-peroxidase method for paraffin-embedded, formalin-fixed tissue sections. Fourteen hyperplastic human tonsils were used, and the results of the binding spectra of each lectin were compared. In general, the binding spectrum detected in the paraffin sections was part of the broader range of affinity obtained in the frozen sections. The lectin receptors on the cell surface were especially affected by formalin fixation. On the other hand, the paraffin sections, because of their superior morphology, showed a better localization of the cytoplasmic reaction product and discriminated the cell types more accurately. Thus, the two tissue preparations are rather complementary. In the tonsil peanut agglutinin (PNA) and periodic acid/Concanavalin A (PA/Con A) proved to be suitable tools for distinguishing exactly between the crypt and the surface epithelium. Ulex europaeus agglutinin I (UEA) is a reliable endothelial marker with a strong affinity to the crypt epithelium. In the frozen sections, PNA regularly stained follicular-centre cells on their cell surface. PNA, Helix pomatia agglutinin (HPA), soybean agglutinin (SBA) and Con A stained the histiocytic population, especially PNA which additionally stained an "asteroid" histiocyte. This cell probably corresponds to the antigen-presenting histiocyte of the T-system.     

On the hemagglutination reaction in methanotrophic bacteria

The reaction of hemagglutination with trypsin-treated rabbit erythrocytes was used to reveal lectins on the cell surface of methanotrophic bacteria and in their culture liquids. By this method, no lectins were detected on the cell surface ofMethylococcus capsulatus IMV B-3001 andMethylomonas rubra IMV B-3075 or in the culture liquid of any of the species studied. With intact cells ofMethylocystis parvus IMV B-3491, the positive hemagglutination reaction observed was nonspecific and most probably occurred due to the high cell surface hydrophobicity characteristic of this species.     

Preparation of apical plasma membranes from cells grown on coverslips. Electron microscopic investigations of the protoplasmic surface

We introduce here a simple method which permits an efficient isolation of apical plasma membranes from tissue culture cells and the electron microscopic examination of their protoplasmic surfaces by use of the platinum/carbon replica technique. Different procedures were tested with regard to the efficiency of isolation and preservation of ultrastructure. Best results were obtained by prestabilization of cell surfaces with low concentrations of tannic acid prior to isolation. To demonstrate the possible applications and versatility of the method, studies were done on virus-infected cells in combination with immunocytochemical labeling. With this model system, we show that it is possible to correlate the structures seen on the cytoplasmic surface of the plasma membrane with the distribution of virus antigens at the cell surface labeled with immunogold markers prior to preparation.     

Growth factor production and autocrine mechanism of cell proliferation regulation in the RPMI-6410t lymphoblastoid line

The human lymphoblastoid B-cell line RPMI-6410t was found to synthesize and secrete into the growth medium a factor necessary to maintain the reproduction of these cells. In the condition of low plating density (concentration 1-1000 cells per ml) cell proliferation can be maintained only in the presence of a definite dose of medium conditioned by 6410t cell growth under high concentration. Using such a medium guaranteed almost 100% cloning efficiency of these cells by the method of limiting dilutions. The cloning of 6410t cells in the presence of feeder cells, such as mouse splenocytes and peritoneal cells, failed. The 6410t cells were shown to bind specifically the growth factor secreted by them, thus suggesting the presence of a growth factor acceptor on their surface. With the help of special selective method some clones were derived which did not secrete growth factor but were likely to have growth factor acceptors on their surface. A comparison of growth properties of clones GF- and GF+ supported the idea of autocrine control of proliferation as one of the mechanisms of malignant cell transformation.     

Effect of Hydrofluoric Acid Etching Time on Titanium Topography,Chemistry, Wettability,and Cell Adhesion

Titanium implant surface etching has proven an effective method to enhance cell attachment. Despite the frequent use of hydrofluoric (HF) acid, many questions remain unresolved, including the optimal etching time and its effect on surface and biological properties. The objective of this study was to investigate the effect of HF acid etching time on Ti topography, surface chemistry, wettability, and cell adhesion. These data are useful to design improved acid treatment and obtain an improved cell response. The surface topography, chemistry, dynamic wetting, and cell adhesiveness of polished Ti surfaces were evaluated after treatment with HF acid solution for 0, 2; 3, 5, 7, or 10 min, revealing a time-dependent effect of HF acid on their topography, chemistry, and wetting. Roughness and wetting increased with longer etching time except at 10 min, when roughness increased but wetness decreased. Skewness became negative after etching and kurtosis tended to 3 with longer etching time. Highest cell adhesion was achieved after 5–7 min of etching time. Wetting and cell adhesion were reduced on the highly rough surfaces obtained after 10-min etching time.     

Single-laser polarization FRET (polFRET) on the cell surface

A new method for the simultaneous detection of rotational mobility and proximity of cell surface receptors is presented based on cell-by-cell basis measurement of polarized fluorescence intensity components of the donor and acceptor of a FRET system. In addition to the FRET efficiency and the donor and acceptor concentrations, the method makes also possible the determination of the rotational characteristics and the associated fraction of the donors (FRET-fraction). The method is illustrated with flow cytometric and rFLIM measurements on donor–acceptor systems comprising fluorescently labeled whole antibodies and their Fab fragments against epitopes of the MHCI and MHCII cell surface receptors on human lymphoblast cells. Fluorescence anisotropy of donor and acceptor and FRET efficiency were measured for samples of different acceptor-to-donor concentration ratios. Acceptor anisotropy proved to be more sensitive than the donor anisotropy for sensing FRET. After determining the rotational constants of the donor-conjugated antibodies by measurements of FRET in the steady state, and by rFLIM as a reference, the associated fractions of the MHCI and MHCII molecules in their clusters were determined. Besides the flow cytometer and the wide-field rFLIM used in this study, the method can be applied also in other devices capable of dual-anisotropy detection.     

Surface-active properties of Candida albicans

Cell surface hydrophobicity may be an important factor contributing to the virulence of Candida yeast cells. Surface hydrophobic and surface polar groups would be required for a yeast cell to act as a surface-active agent. In this report, the surface activities of whole yeast cells were measured. Yeast cells added at 10(8)/ml reduced the surface tension (gamma s) of saline by 20% as determined by the du Nouy method. A 1% suspension of yeast cell wall fragments reduced gamma s of saline by 36%. Whole yeast cells caused a reduction in interfacial tension (gamma I) between hexadecane and saline. The reduction of gamma I was proportional to the surface hydrophobicity of the yeasts. Yeast cells grown in glucose as the sole carbon source (thus possessing a relatively more hydrophilic cell surface) reduced gamma I by 30%, whereas yeast cells grown in hexadecane (thus possessing a more hydrophobic cell surface) reduced gamma I by 41%. The reduction of gamma I was reversed upon the addition of a strong surfactant. It was also demonstrated that yeast cells blended with nonionic surfactants during growth in a glucose broth in order to change their cell surface hydrophobicity adhered to solid surfaces in direct proportion to their cell surface hydrophobicity. Thus, the surface-active properties of Candida yeast cells may significantly contribute to the accumulation of yeast cells at various biological interfaces such as liquid-solid, liquid-liquid, and liquid-air, leading to their eventual adhesion to solid or tissue surfaces.     

Surface-active properties of Candida albicans.

Cell surface hydrophobicity may be an important factor contributing to the virulence of Candida yeast cells. Surface hydrophobic and surface polar groups would be required for a yeast cell to act as a surface-active agent. In this report, the surface activities of whole yeast cells were measured. Yeast cells added at 10(8)/ml reduced the surface tension (gamma s) of saline by 20% as determined by the du Nouy method. A 1% suspension of yeast cell wall fragments reduced gamma s of saline by 36%. Whole yeast cells caused a reduction in interfacial tension (gamma I) between hexadecane and saline. The reduction of gamma I was proportional to the surface hydrophobicity of the yeasts. Yeast cells grown in glucose as the sole carbon source (thus possessing a relatively more hydrophilic cell surface) reduced gamma I by 30%, whereas yeast cells grown in hexadecane (thus possessing a more hydrophobic cell surface) reduced gamma I by 41%. The reduction of gamma I was reversed upon the addition of a strong surfactant. It was also demonstrated that yeast cells blended with nonionic surfactants during growth in a glucose broth in order to change their cell surface hydrophobicity adhered to solid surfaces in direct proportion to their cell surface hydrophobicity. Thus, the surface-active properties of Candida yeast cells may significantly contribute to the accumulation of yeast cells at various biological interfaces such as liquid-solid, liquid-liquid, and liquid-air, leading to their eventual adhesion to solid or tissue surfaces.     

Retention of the glycocalyx after cell detachment by EGTA.

Chinese hamster ovary cells were examined ultrastructurally following several detachment procedures. Alterations in the surface glycoproteins were observed by using ruthenium red in the fixation procedure. Trypsin removed a major portion of the cells glycocalyx and formed spherical cellular configurations. EGTA detached cells were also spherical, however, their glycocalyx appeared to remain although redistributed over the cell surface. Kinetic studies showed no alterations in subsequent population doubling times following either detachment procedure. EGTA may thus represent the current method of choice for cell detachment if preservation of the surface glycoproteins is of interest.     

Electrophoretic mobility of mouse cells and homologous isolated nuclei

The electrophoretic mobilities of Ehrlich ascites, sarcoma 37 ascites, mouse liver cells and their isolated nuclei were measured under similar environmental conditions. No differences in mobility were detected between cells and homologous nuclei from the same cell population and it was concluded that their surface charge densities were probably the same. The effect of neuraminidase on Ehrlich ascites and liver cells and nuclei was also determined; neuraminidase reduced the mobility of Ehrlich ascites cell nuclei as well as cells. The reduction in mobility of cells and nuclei prepared by a sucrose method was the same; however, the reduction in mobility of citric acid prepared nuclei was less than that of citric acid treated cells. The reduction in mobility of both liver cells and nuclei was small or insignificant. It is suggested that although cells and nuclei have similar electrophoretic mobilities, possibly different groups contribute to their surface charge.     

Selective isolation of bacteria from soil with hydrophobic materials

Bacterial strains having a hydrophobic cell surface have often been considered as degraders of hydrophobic organic pollutants in soil. In this study, bacterial strains were isolated using hydrophobic materials from 12 soil samples, and their cell surface hydrophobicity was determined by evaluating their adherence to n-hexane. Bacterial strains isolated using polytetrafluoroethylene (PTFE) membrane were more hydrophobic on an average than those isolated with styrene–divinylbenzene (DVB) particles or octadecylsilyl silica gel (ODS) particles. Strains closely related to Burkholderia cepacia could be selectively isolated using the PTFE membrane; those closely related to Ralstonia pickettii, using ODS and DVB particles; and those closely related to B. fungorum, using DVB. These results indicate that bacterial strains having a hydrophobic cell surface or within certain phyla can be selectively isolated from soils using hydrophobic materials, and that this isolation method would be useful for collecting candidates for bioremediation of hydrophobic pollutants.     

Cell–surface interactions involving immobilized magnetite nanoparticles on flat magnetic substrates

A new method to affect cells by cell–surface interaction is introduced. Biocompatible magnetic nanobeads are deposited onto a biocompatible magnetic thin layer. The particles are composed of small magnetite crystals embedded in a matrix which can be functionalized by different molecules, proteins or growth factors. The magnetic interaction between surface and beads prevents endocytosis if the setup is utilized for cell culturing. The force acting between particles and magnetic layer is calculated by a magnetostatic approach. Biocompatibility is ensured by using garnet layers which turned out to be nontoxic and stable under culturing conditions. The garnet thin films exhibit spatially and temporally variable magnetic domain configurations in changing external magnetic fields and depending on their thermal pretreatment. Several patterns and bead deposition methods as well as the cell–surface interactions were analyzed. In some cases the cells show directed growth. Theoretical considerations explaining particular cell behavior on this magnetic material involve calculations of cell growth on elastic substrates and bending of cell membranes.     

Potential of immunogold-silver staining for the study of leukocyte subpopulations as defined by monoclonal antibodies

The potential of immunogold-silver staining for study of leukocyte subpopulations, as defined by monoclonal antibodies in cell suspensions, was examined. The cells were labeled in suspension as described for immunogold staining. Cytocentrifuge preparations of the suspensions were then immersed in a physical developer. By light microscopy, cells reacting with the monoclonal antibodies showed dark granules on their surface membrane. The morphology of the cells, as revealed by a panoptic counterstain, was comparable with that seen in routine cell smears for differential counts. The numbers of T-cells, T-helper/inducer cells, and T-cytotoxic/suppressor cells counted by this method in normal peripheral blood were nearly identical to those identified by immunogold staining and immunofluorescence microscopy in the same cell suspensions. The good morphological delineation also made possible rapid and accurate identification of particular leukocyte subsets in complex cell suspensions. Atypical lymphocytes from patients with infectious mononucleosis displayed the surface phenotype of activated T-cytotoxic/suppressor cells. Different maturation stages of neoplastic cells in patients with acute myeloid leukemia showed differences in surface antigen expression. Immunological detection of cell surface antigens could be combined with cytochemical staining of intracellular enzymatic activities. Finally, the labeling could be performed on cells prefixed on glass slides.     

Effect of cycloheximide and renewal of phosphorus supply on surface acid phosphatase activity of phosphorus deficient tomato roots

Changes in surface phosphatase activity of tomato root ( Lycopersicon esculentum L. cv. Marmande) have been studied in relation to its P status. Experiments were performed either with excised roots cultured in vitro or with entire plants (split root method) grown in various conditions (P deficiency, renewal of P supply). In some experiments different parts of the root were separated according to their P status during their primary growth.
The surface phosphatase in different root parts depends first of all on P status during their primary growth. Moreover the cell wall phosphatase of tomato root is stable in vivo . Thus the changes in the surface phosphatase of a root system result mainly from the growth of new roots, which bear an enzyme activity that is either high if they are P deficient or low if they are P provided. The control of the cell wall phosphatase synthesis appears to be a repression-derepression process mediated by the root cell concentration in some P compound, probably orthophosphate. Cycloheximide stops or alters the growth of excised roots, so that this inhibitor was found unsuitable to study the synthesis of cell wall phosphatase under in vivo conditions. Split root experiment shows that the increase in surface phosphatase activity may occur locally, i.e. only in the parts of the root system which are P deficient. Agricultural and ecological aspects of these findings are pointed out.