Some speculations on the rate of adhesion of cells to coverslips

Suspensions of mouse tumour cells were cultured over glass coverslips, and their rate of adhesion to the coverslips measured. The interaction energies between the cell and coverslip surfaces were computed according to DLVO-theory, on the basis of experimentally determined parameters, for a wide range of values of Hamaker's coefficients. The secondary attractive minimum was computed to occur when the two surfaces were separated by 40–85 Å. It was observed that 50% of the cells adhered to the coverslips after 30 minutes, although it was calculated that they could gravitate to the position of the secondary minima in approximately 5 minutes. We have discussed this delay of 25 minutes in terms of the mechanisms of cell adhesion to the coverslips.     

Epithelial macrophages secrete a deactivating factor for superoxide release

The release of superoxide anion (O2-) by inflammatory macrophages, multinucleated giant cells, and epithelioid cells, obtained by the insertion of round glass coverslips into the subcutaneous tissue of mice, was investigated. O2- was shown to be spontaneously released by cells on the surface of glass coverslips implanted up to 7 days, but not by cells obtained 14 or 21 days after coverslip implantation. The former showed increased O2- release when stimulated by phorbol myristate acetate, whereas cells harvested after 14 or 21 days implantation did not. The induction of delayed type hypersensitivity around coverslips implanted for 5 days increased spontaneous O2- release by these cells by 40%. On the other hand, when the same protocol was used with coverslips implanted for 14 days, O2- release was not detected. These results were viewed in regard to the composition of the cell population at each time point. When coverslips were removed after 14 days of implantation and the cells incubated for 30 minutes in vitro, the medium so conditioned inhibited O2- release by cells of 5 day old preparations. This indicates the release by cells on the longer term coverslips of a substance that inhibits O2- production by cells of coverslips implanted for 5 days only. This inhibitory activity could be suppressed by treating the conditioned medium with proteases. The factor was, however, heat stable and exerted its effects even when the test cells were exposed to phorbol myristate acetate.     

Coating of coverslips with glow-discharged carbon promotes cell attachment and spreading probably due to carboxylic groups

BACKGROUND: For high-resolution microscopy, cells have to be analyzed through thin glass coverslips. Therefore, it is necessary to culture cells on coverslips for preservation of cell morphology. We found cell attachment and spreading to be relatively slow processes, even when cells were plated on coated coverslips. This slowness presents a problem, particularly when synchronized cell populations are used. METHODS: In this paper, we present a method that is based on glow-discharged carbon coating of coverslips which promotes rapid attachment and spreading of cells, enabling rapid analysis of cells after plating. Results obtained with carbon-coated coverslips were compared with those of other types of coating. Two fibroblast lines, an epithelial cell line, and a carcinoma cell line were tested. RESULTS AND CONCLUSIONS: All cell lines showed a rapid adhesion on carbon-coated coverslips. With fibroblasts we found the carbon coating to be superior to other coatings tested, mainly because the carbon did not influence cell morphology. Using synchronized or irradiated cells produced similar results. The superior performance of carbon coating is probably due to carboxylic groups on the glow-discharged carbon layer. The carbon layer does not interfere with microscopy or immunocytochemical staining procedures.     

The effect of flow on the interaction of isolated megakaryocytes with subendothelial extracellular matrix.

We have previously shown that human, guinea pig, or rat megakaryocytes, incubated under static conditions on an extracellular matrix (ECM) produced by endothelial cells, readily adhered to the matrix and underwent platelet-like shape change and thromboxane A2 secretion. We have now exposed megakaryocytes to ECM in a perfusion system similar to that used to study platelets circulated over aortic subendothelium. We used a continuous flow circuit incorporating a parallel plate perfusion chamber. Megakaryocytes were isolated to high purity from guinea pig marrow by centrifugal elutriation and velocity sedimentation. The cells were introduced into the flowing medium while the surface of an ECM-coated coverslip mounted in the chamber was observed continuously by phase-contrast video microscopy for up to 18 hours. Megakaryocytes from the flowing suspension started to adhere to the ECM within seconds. Significant adhesion occurred over a range of shear rates, from 10 to 190 seconds-1, did not appear above 300 seconds-1 and was greatest at a shear rate of 60 seconds-1. Adhesion to the ECM was specific, since there was no adherence to glass coverslips, glutaraldehyde-fixed ECM-coated coverslips, or to endothelial cells cultured on ECM-coated coverslips. At low shear rates large aggregates of megakaryocytes formed on the ECM surface; these could be detached and washed away by higher shear forces. Megakaryocytes thus acquire, even before platelet formation, an adhesive capacity similar to that of platelets. In addition, a significant fraction of the adherent megakaryocytes underwent elongation and pseudopod formation similar to that seen in marrow sinusoids.     

Adhesion of biodegradative anaerobic bacteria to solid surfaces.

In order to exploit the ability of anaerobic bacteria to degrade certain contaminants for bioremediation of polluted subsurface environments, we need to understand the mechanisms by which such bacteria partition between aqueous and solid phases, as well as the environmental conditions that influence partitioning. We studied four strictly anaerobic bacteria, Desulfomonile tiedjei, Syntrophomonas wolfei, Syntrophobacter wolinii, and Desulfovibrio sp. strain G11, which theoretically together can constitute a tetrachloroethylene- and trichloroethylene-dechlorinating consortium. Adhesion of these organisms was evaluated by microscopic determination of the numbers of cells that attached to glass coverslips exposed to cell suspensions under anaerobic conditions. We studied the effects of the growth phase of the organisms on adhesion, as well as the influence of electrostatic and hydrophobic properties of the substratum. Results indicate that S. wolfei adheres in considerably higher numbers to glass surfaces than the other three organisms. Starvation greatly decreases adhesion of S. wolfei and Desulfovibrio sp. strain G11 but seems to have less of an effect on the adhesion of the other bacteria. The presence of Fe(3+) on the substratum, which would be electropositive, significantly increased the adhesion of S. wolfei, whereas the presence of silicon hydrophobic groups decreased the numbers of attached cells of all species. Measurements of transport of cells through hydrophobic-interaction and electrostatic-interaction columns indicated that all four species had negatively charged cell surfaces and that D. tiedjei and Desulfovibrio sp. strain G11 possessed some hydrophobic cell surface properties. These findings are an early step toward understanding the dynamic attachment of anaerobic bacteria in anoxic environments.     

Adhesive properties of sea cucumber coelomoyctes

Summary— The adhesive properties of the coelomocytes of the sea cucumber, Holothuria polii, have been investigated by studying their ability to attach to glass coverslips in vitro, and their morphology examined by scanning electron microscopy. Both amoeboyctes and spherule cells in cell suspensions attached themselves to glass coverslips, but spreading activity was restricted to an amoebocyte subset which assumed an extremely flattened morphology. Coelomocyte adhesion was a time- and temperature-dependent phenomenon and required cations for attachment to the glass surface. Mg²⁺ ions were more effective than Ca²⁺ in facilitating cell binding. The addition of potassium cyanide or sodium azide to the cell suspension did not inhibit amoebocyte attachment but vinblastine did. Cytochalasin B had no effect. Cell adhesiveness was greatly enhanced with both coelomic fluid and purified 200-kDa coelomocyte-aggregating factor.     

Development of an artificial neuronal network with post-mitotic rat fetal hippocampal cells by polyethylenimine

The selection of appropriate surface materials that promote cellular adhesion and growth is an important consideration when designing a simplified neuronal network in vitro. In the past, extracellular matrix proteins such as laminin (LN) or positively charged substances such as poly-l-lysine (PLL) have been used. In this study, we examined the ability of another positively charged polymer, polyethyleneimine (PEI), to promote neuronal adhesion, growth and the formation of a functional neuronal network in vitro. PEI, PLL and LN were used to produce grid-shape patterns on glass coverslips by micro-contact printing. Post-mitotic neurons from the rat fetal hippocampus were cultured on the different polymers and the viability and morphology of these neurons under serum-free culture conditions were observed using fluorescent microscopy and atomic force microscopy (AFM). We show that neurons cultured on the PEI- and PLL-coated surfaces adhered to and extended neurites along the grid-shape patterns, whereas neurons cultured on the LN-coated coverslips clustered into clumps of cells. In addition, we found that the neurons on the PEI and PLL-coated grids survived for more than 2 weeks in serum-free conditions, whereas most neurons cultured on the LN-coated grids died after 1 week. Using AFM, we observed some neurosynapse-like structures near the neuronal soma on PEI-coated coverslips. These findings indicate that PEI is a suitable surface for establishing a functional neuronal network in vitro.     

Photoengraving of coverslips and slides to facilitate monitoring of micromanipulated cells or chromosome spreads

A simple photolithographic technique has been developed which can be used to produce microscopic grid patterns on glass coverslips. The grid pattern is first photo-reduced onto film, and the resulting photographic negative is then used as a mask. A glass slide or coverslip, coated with a layer of photoresist, is then exposed to tungsten light through the mask. After developing and etching, the grid pattern is transferred permanently onto glass. This simple and rapid procedure allows one to mass-produce very small, high resolution grids which are useful for monitoring individual microinjected cells or chromosomal spreads under the microscope.     

Fluoroplastic Coverslips for Long-Term Nerve Tissue Culture

Fluoroplastics (Aclar and Teflon FEP) were tested for their suitability as coverslip material for nerve tissue cultures. Coverslips prepared from these plastics have a higher transparency over the near-ultraviolet, visible, and infrared regions of the spectrum than glass coverslips, possess exceptional physical and chemical stability, and provide service-ability over a wide temperature range ≥ -320 to 390 F. For nerve tissue culture, circular 22 mm coverslips were cut from 5 mil sheets of Aclar 22A and 33C, and Teflon FEP 100A, cleaned in concentrated HNO₃, rinsed, sterilized in 85% ethyl alcohol, dried, and coated with collagen. Peripheral and central nervous system cultures from fetal rats and newborn mice were set up on these as well as on glass coverslips, and carried in Maximow double coverslip assemblies. Over a 2 mo period of cultivation, neuronal maturation, tissue organization and fiber myelination occurred on plastic coverslips in essentially the same manner as in sister cultures maintained on glass coverslips. Cultures fixed and embedded in Epon for electron microscopy directly on Aclar or Teflon 100A coverslips were easily separated from the coverslip. Some difficulty was encountered with retraction of the collagen coat from Teflon 100A coverslips, however, and Aclar 22A coverslips were warped by some of the reagents used in embedding for electron microscopy. Aclar 33C was not adversely affected by these reagents, and provided the most suitable base for collagenization and culture maintenance. A variety of cells grew satisfactorily on either the bare or the collagenized surface of this plastic, and separated easily from it following embedment in Epon.     

Effect of cell substratum on lateral mobility of lipids in the plasma membrane of vascular endothelial cells

Bovine vascular endothelial cells can be maintained in a highly differentiated state in vitro, either by the addition of fibroblast growth factor (FGF) to the culture medium or by plating the cells on extracellular matrix (ECM)-coated dishes. Under these conditions the cells proliferate actively and at confluence form a tightly packed monolayer composed of nonoverlapping polarized cells. A fluorescence recovery after photobleaching method was used to determine the lateral mobility coefficient D of the lipophilic fluorescent probe, 5N-(hexadecanoyl)-aminofluorescein (HEDAF), in the basal and apical plasma membranes of endothelial cells under various culture conditions (cells on glass coverslips in the presence or absence of FGF, or cells plated on ECM in the exponential growth phase or at confluence). A heterogeneous distribution of lateral diffusion coefficients D was found in a given cell population. Nevertheless, for the basal membrane, a "mean" D value close to 2.0 x 10(-9) cm2/s was found for all the culture conditions. The "mean" D value of HEDAF in the apical pole was slightly higher when sparse cells were exposed to FGF (D = 2.2 x 10(-9) cm2/s) and was further enhanced when cells were growing or confluent on ECM-coated coverslips (D = 2.7 x 10(-9) cm2/s). On the other hand, when the cells were maintained in the absence of FGF on glass coverslips, similar "mean" D values were found in both cell poles (D = 2.0 x 10(-9) cm2/s). These results show that lateral mobility of lipids in endothelial plasmalemma varies in response to external factors such as FGF and the ECM.     

Quantitative studies of increased cell-to-substratum adhesion at low temperature

The cell-to-substratum adhesion of an established epithelial cell line cultured for 24 h on glass coverslips was determined at 4 degrees C, 8 degrees C and 37 degrees C using a miniaturised parallel-plate shearing apparatus. The measurements of the minimum shear necessary to dislodge the cells (minimum distraction force, MDF) demonstrated a three- to fourfold increase in the adhesion of the cells at 4 degrees C (6.17 Pa) compared to that at 37 degrees C (1.36 Pa). At 8 degrees C the MDF was 2.31 pascals. Part of the adhesion was resistant to mild trypsinisation. Trypsin-resistant adhesion (TRA) was stabilised by low temperature, and by treatment with concanavalin A (50 micrograms ml-1) or colchicine (200-400 microM). The effects of con A (140 micrograms ml-1) and low temperature (4 degrees C) were additive, giving a combined MDF of greater than 9.27 pascals. On the basis of their different temperature and protease susceptibility it is suggested that trypsin-sensitive adhesion (TSA) and TRA represent separate functional classes of cell-to-substratum attachment corresponding to 'frictional' and 'tractional' adhesion, respectively.     

Sample preparation and imaging of erythrocyte cytoskeleton with the atomic force microscopy

A novel method for the covalent attachment of erythrocytes to glass microscope coverslips that can be used to image intact cells and the cytoplasmic side of the cell membrane with either solid or liquid mode atomic force microscopy (AFM) is described. The strong binding of cells to the glass surface is achieved by the interaction of cell membrane carbohydrates to lectin, which is bound to N-5-azido-2-nitrobenzoyloxysuccinimide (ANBNOS)-coated coverslips (1). The effectiveness of this method is compared with the other commonly used methods of immobilizing intact erythrocytes on glass coverslips for AFM observations. Experimental conditions of AFM imaging of biologic tissue are discussed, and typical topographies of the extracellular and the cytoplasmic surfaces of the plasma membrane in the dry state and in the liquid state are presented. Comparison of the spectrin network of cell age-separated erythrocytes has demonstrated significant loss in the network order in older erythrocytes. The changes are quantitatively described using the pixel height histogram and window size grain analysis.     

Influence of polylysine on adhesion of fibroblasts to glass substrates visualized by total internal reflection microscopy

The cell adhesion topography of mouse fibroblasts growing on glass substrates has been investigated. In order to compare cell adhesion on covered and uncovered glass, substrates were partly exposed to a solution with 0.1 mg/ml polylysine (300 kDa) for 15 min before incubation with cell suspension. After cultivation for 1, 3, 6, and 24 h their adhesion was visualised by total internal reflection microscopy. In the presence of polylysine, cells incubated for 1 h were strongly attracted to the substrate, leading to a typical cell adhesion topography characterised by round concavities under the ventral cell membrane with an approximate diameter of 1 μm. The cavity-surrounding rims were tightly bound to the glass surface. During further cell cultivation, the topography changed into a well-organised adhesion pattern with focal contact areas on the periphery of the cells. In contrast to the polylysine-mediated adhesion, cells growing on untreated surfaces did not exhibit the cavity-like topography at any stage of cultivation, but a more point spread adhesion with a dense clustering of contact-forming areas.     

Adhesion of Biodegradative Anaerobic Bacteria to Solid Surfaces

In order to exploit the ability of anaerobic bacteria to degrade certain contaminants for bioremediation of polluted subsurface environments, we need to understand the mechanisms by which such bacteria partition between aqueous and solid phases, as well as the environmental conditions that influence partitioning. We studied four strictly anaerobic bacteria, Desulfomonile tiedjei, Syntrophomonas wolfei, Syntrophobacter wolinii, and Desulfovibrio sp. strain G11, which theoretically together can constitute a tetrachloroethylene- and trichloroethylene-dechlorinating consortium. Adhesion of these organisms was evaluated by microscopic determination of the numbers of cells that attached to glass coverslips exposed to cell suspensions under anaerobic conditions. We studied the effects of the growth phase of the organisms on adhesion, as well as the influence of electrostatic and hydrophobic properties of the substratum. Results indicate that S. wolfei adheres in considerably higher numbers to glass surfaces than the other three organisms. Starvation greatly decreases adhesion of S. wolfei and Desulfovibrio sp. strain G11 but seems to have less of an effect on the adhesion of the other bacteria. The presence of Fe³⁺ on the substratum, which would be electropositive, significantly increased the adhesion of S. wolfei, whereas the presence of silicon hydrophobic groups decreased the numbers of attached cells of all species. Measurements of transport of cells through hydrophobic-interaction and electrostatic-interaction columns indicated that all four species had negatively charged cell surfaces and that D. tiedjei and Desulfovibrio sp. strain G11 possessed some hydrophobic cell surface properties. These findings are an early step toward understanding the dynamic attachment of anaerobic bacteria in anoxic environments.     

Morphological responses of dissociated sponge cells to different organic substrata

To study interactions between sponge cells and components of the extracellular matrix (ECM), cells of the calcareous sponge Clathrina cerebrum were investigated in vitro by scanning electron microscopy. Cells were settled on glass coverslips, used as controls, and on coverslips coated with various ECM components (laminin, collagens and fibronectin), and with an adhesive substance (polylysine). Cells tended to conserve a rounded shape, producing thin, stiff processes (scleropodia) and lamellipodia, whose shape and extension varied according to the substrata. Spreading was observed only on polylysine, inducing cells to assume a fibroblast-like aspect. On laminin, cell adhesion was assured only by scleropodia. On fibronectin, scleropodia and lamellipodia were present, but reduced in size and length. On collagens, laminar processes occurred among prevailing scleropodia. Measurements of cell area and perimeter allowed statistical comparison of substrata, on the basis of their induction of cell flattening and protuberance formation. In summary, sponge cells were found to modulate their morphology in response to the external environment, expressing features for dynamic activities most fully in the presence of substances close to their natural ECM constituents. These results are discussed in the context of tissue rearrangement as a basic adaptation occurring throughout the life span of these organisms.     

The anti-invasive flavonoid (+)-catechin binds to laminin and abrogates the effect of laminin on cell morphology and adhesion

To study the effect of the flavonoid (+)-catechin on cell-matrix interactions two cell types with a different morphology on and adhesion to laminin were used. MO4 virally transformed fetal mouse cells adhere and spread when cultured on top of laminin-coated coverslips or on human amnion basement membrane. M5076 mouse reticulum cell sarcoma cells poorly adhere to these substrates and remain round. Both cell types are invasive in confronting cultures with embryonic chick heart fragments. (+)-Catechin binds to laminin in a pH-dependent way. Pretreatment of laminin-coated coverslips or amnion basement membrane with 0.5 mM (+)-catechin abrogates the effect of laminin on cell morphology and adhesion. MO4 cells do not adhere to the pretreated substrates and remain round, while M5076 cells now adhere and spread. (+)-Catechin inhibits the invasion of MO4 cells but not of M5076 cells into embryonic chick heart in vitro. We speculate that the anti-invasive activity of the flavonoid to MO4 cells is the result of its interference with MO4 cell adhesion to laminin. Invasion of M5076 cells does not imply adhesion to and spreading on laminin.     

High-resolution three-dimensional views of membrane-associated clathrin and cytoskeleton in critical-point-dried macrophages

We obtained high-resolution topographical information about the distribution of clathrin and cytoskeletal filaments on cytoplasmic membrane surfaces of macrophages spreading onto glass coverslips by both critical-point drying of broken-open cells and preparation of rotary platinum replicas. Irregular patches of the adherent ventral surface of the plasma membrane were exposed in these cells, and large areas of these exposed membranes were covered with clathrin-coated patches, pits, and vesicles. Various amounts of cytoskeleton were attached to the plasma membranes of these spreading cells, either as distinct starlike foci, or as individual filaments and bundles radiating out from the cytoskeletal meshwork. In newly adherent cells a well developed Golgi-GERL complex, characterized by smooth, dish-like cisternae associated with rough endoplasmic reticulum, was observed. There were many coated vesicles budding off from the Golgi cisternae, and these were predominantly of the large type (150 nm) usually associated with the plasma membrane. In critical-point-dried samples, both cytoskeleton and membranes were preserved in detail comparable to that of quick-frozen samples, after appropriate fixation. Rotary replication of critical-point-dried cells provides a rapid, easily controlled, and generally easy to perform method for obtaining samples of exposed membrane large enough to permit quantification of membrane- associated clathrin and cytoskeleton under various experimental conditions.     

Effect of extracellular matrix proteins on in vitro testosterone production by rat Leydig cells

The aim of this study was to detect the effect of extracellular matrix (ECM) proteins on rat Leydig cell shape, adhesion, expression of integrin subunits and testosterone production, in vitro. Leydig cells isolated from adult rats were cultured on plates uncoated or coated with different concentrations of laminin-1, fibronectin, or type IV collagen in the presence or absence of hCG for 3 or 24 hr. A significant increase of cell adhesion and of alpha3, alpha5, and beta1 integrin subunit expression was observed when cells were cultured on ECM proteins, compared to those grown on uncoated plates. Leydig cells cultured on glass coverslips coated with ECM proteins for 24 hr exhibited elongated shapes with long cell processes (spreading), while cells cultured on uncoated plates showed few cell processes. A significant decrease in testosterone production was observed when basal and hCG-stimulated Leydig cells were cultured for 3 or 24 hr on plates coated with type IV collagen (12 and 24 microg/cm(2)) compared to uncoated plates. A significant though a slighter decrease in testosterone production was also observed in cells cultured on plates coated with fibronectin (12 and 24 microg/cm(2)), compared to uncoated plates. Laminin-1 did not modify testosterone production under basal or hCG stimulated conditions. These results suggest that ECM proteins are able to modulate Leydig cell steroidogenesis, in vitro.     

A high-yield technique for preparing cells fixed in suspension for scanning electron microscopy

Human leukocytes fixed in suspension were allowed to settle onto poly-L-lysine-coated glass coverslips and prepared for observation with the scanning electron microscope (SEM). The coverslips were dehydrated in ethanol, critical point dried with CO2, and coated with gold-palladium. With the aid of a locator grid, several fields were photographed with light microscopy after the cells had settled onto the poly-L-lysine-coated coverslips and again after completion of the processing before SEM observation. Quantitative comparison of the number of cells present after settling with the number retained for final viewing with the SEM revealed a cell yield approaching 100%. This simple, reproducible, high-yield technique for processing cells fixed in suspension for SEM prevents changes in surface architecture induced by collecting live cells onto various substrates before fixation and also avoids potentially selective cell losses. Such a technique should allow quantitative correlations between SEM and other morphological and functional parameters.     

Phosphatidylethanolamine Deficiency Impairs Escherichia coli Adhesion by Downregulating Lipopolysaccharide Synthesis,Which is Reversible by High Galactose/Lactose Cultivation

As the initiation step of bacterial infection or biofouling, bacterial adhesion on cells or substrates is generally an optimal target for antibacterial design. Phosphatidylethanolamine (PE) is the principal phospholipid in bacteria, and its function in bacterial adhesion remains unclear. In this study, four E. coli strains including two PE-deficient mutants (PE^?PC^? and PE^?PC^+?strains) and two PE-containing wild-type controls (PE?⁺?PC^? strains) were recruited to investigate the influence of PE deficiency on bacterial adhesion. We found that PE deficiency could impair E. coli adhesion on macrophages (human THP-1-derived and mouse RAW264.7 macrophages) or glass coverslips by downregulating lipopolysaccharide (LPS) biosynthesis, which could be reversible by high galactose/lactose but not glucose cultivation. The data imply that PE play important role in bacterial adhesion probably via affecting LPS biosynthesis and suggest that targeting PE biosynthesis is also a potential antibacterial strategy.