Determination of binding strength and kinetics of binding initiation. A model study made on the adhesive properties of P388D1 macrophage-like cells

The adhesive properties of the mouse P388D1 macrophage-like line were explored. Cells were deposited in glass capillary tubes, and the kinetics of adhesion and spreading were studied. Binding involved the cell metabolism since it was decreased by cold, azide, or a divalent cation chelator. Glass-adherent cells were subjected to calibrated laminar shear flows with a highly viscous dextran solution. A tangential force of about 5 X 10(-3) dyn/cell was required to achieve substantial detachment. The duration of application of the shearing force strongly influenced cell-substrate separation when this was varied from 1-10 s. Further, this treatment resulted in marked cell deformation, with the appearance of an elongated shape. Hence, cell-substrate separation is a progressive process, and binding strength is expected to be influenced by cell deformability. The minimum time required for adhesion was also investigated by making cells adhere under flow conditions. The maximum flow rate compatible with adhesion was about 1000-fold lower than that required to detach glass-bound cells. A simple model was devised to provide a quantitative interpretation for the experimental results of kinetic studies. It is concluded that cell-to-glass adhesion required a cell-substrate contact longer than a few seconds. This first step of adhesion was rapidly followed by a large (about 1000-fold) increase of adhesion strength. It is therefore emphasized that adhesion is heavily dependent on the duration of cell-to-cell encounter, as well as the force used to remove so-called unbound cells.     

Cell-substrate separation: effect of applied force and temperature

We measure the change in cell-substrate separation in response to an upward force by combining two relatively new techniques, Electric Cell-substrate Impedance Sensing (ECIS) to measure average cell-substrate separation, and collagen-coated magnetic beads to apply force to the top (dorsal) surface of cells. The collagen-coated ferric oxide beads attach to integrin receptors in the dorsal surfaces of osteoblastlike ROS 17/2.8 cells. Magnetic force is controlled by the position and the number of permanent magnets, applying an average 320 or 560 pN per cell. Comparing model calculations with experimental impedance data, the junctional resistivity of the cell layer and the average distance between the lower (ventral) cell surface and substrate can be determined. The ECIS analysis shows that these forces produce an increase in the distance between the ventral cell surface and the substrate that is in the range of 10 to 25%. At temperatures of 4°, 22° and 37 °C, the measured cell surface-substrate distances without magnetic beads are 84 ± 4, 45 ± 2 and 38 ± 2 nm. The force-induced changes at 22° are 11 ± 3 and 21 ± 3 nm for 320 and 560 pN, and at 37° they are 5 ± 2 and 9 ± 2 nm. The resulting cell-substrate spring constants at 22° and 37° are thus about 28 and 63 pN nm^–1 (dyne cm^–1). Using a reasonable range for the number for individual integrin-ligand adhesion bonds gives a range for the spring constant of the individual adhesion bond of from about 10^–3 to 10^–1 pN nm^–1. These data also provide evidence that the number of adhesion bonds per cell increases with temperature. Received: 20 June 1997 / Accepted: 24 August 1997     

A numerical analysis of forces exerted by laminar flow on spreading cells in a parallel plate flow chamber assay

Exposure of spreading anchorage-dependent cells to laminar flow is a common technique to measure the strength of cell adhesion. Since cells protrude into the flow stream, the force exerted by the fluid on the cells is a function of cell shape. To assess the relationship between cell shape and the hydrodynamic force on adherent cells, we obtained numerical solutions of the velocity and stress fields around bovine aortic endothelial cells during various stages of spreading and calculated the force required to detach the cells. Morphometric parameters were obtained from light and scanning electron microscopy measurements. Cells were assumed to have a constant volume, but the surface area increased during spreading until the membrane was stretched taut. Two-dimensional models of steady flow were generated using the software packages ANSYS (mesh generation) and FIDAP (problem solution). The validity of the numerical results was tested by comparison with published results for a semicircle in contact with the surface. The drag force and torque were greatest for round cells making initial contact with the surface. During spreading, the drag force and torque declined by factors of 2 and 20, respectively. The calculated forces and moments were used in adhesion models to predict the wall shear stress at which the cells detached. Based upon published values for the bond force and receptor number, round cells should detach at shear stresses between 2.5 and 6 dyn/cm(2), whereas substantially higher stresses are needed to detach spreading and fully spread cells. Results from the simulations indicate that (1) the drag force varies little with cell shape whereas the torque is very sensitive to cell shape, and (2) the increase in the strength of adhesion during spreading is due to increased contact area and receptor densities within the contact area. (c) 1993 John Wiley & Sons, Inc.     

Estimating the efficiency of cell capture and arrest in flow chambers: study of neutrophil binding via E-selectin and ICAM-1

A mathematical model was developed to quantify the efficiency of cell-substrate attachment in the parallel-plate flow chamber. The model decouples the physical features of the system that affect cell-substrate collision rates from the biological features that influence cellular adhesivity. Thus, experimental data on cell rolling and adhesion density are converted into "frequency" parameters that quantify the "efficiency" with which cells in the flow chamber progress from the free stream to rolling, and transition from rolling to firm arrest. The model was partially validated by comparing simulation results with experiments where neutrophils rolled and adhered onto substrates composed of cotransfected cells bearing E-selectin and intercellular adhesion molecule-1 (ICAM-1). Results suggest that: 1) Neutrophils contact the E-selectin substrate on average for 4-8.5s before tethering. This contact duration is insensitive to applied shear stress. 2) At 2 dyn/cm(2), approximately 28% of the collisions between the cells and substrate result in primary capture. Also, approximately 5-7% of collisions between neutrophils in the free stream and previously recruited neutrophils bound on the substrate result in secondary capture. These percentages were higher at lower shears. 3) An adherent cell may influence the flow streams in its vicinity up to a distance of 2.5 cell diameters away. 4) Our estimates of selectin on-rate in cellular systems compare favorably with data from reconstituted systems with immobilized soluble E-selectin. In magnitude, the observed on-rates occur in the order, L-selectin > P-selectin > E-selectin.     

Electrically measuring viscoelastic parameters of adherent cell layers under controlled magnetic forces

Electrical cell-substrate impedance sensing (ECIS) was used to measure the time-dependence and frequency-dependence of impedance for current flowing underneath and between cells. Osteosarcoma cells with a topology similar to a short cylinder (coin-like) surmounted by a dome were used in this study. Application of a small step increase in net vertical stress to the cells (4 and 7 dyn/cm²), via magnetic beads bound to the dorsal (upper) surface, causes an increase in cell body height and an increase in cell-cell separation, as well as stretching of the cell-substrate adhesion bonds. This results in a fast drop in measured resistance (less than 2 s), followed by a slower change with a time constant of 60–150 s. This time constant is about 1.5 times longer at 22 °C than that at 37 °C; it also increases with applied stress. Our frequency scan data, as well as our data for the time course of resistance and capacitance, show that the fast change is associated with both the under-the-cells and between-the-cells resistance. The slower change in resistance mainly reflects the between-the-cells resistance. To obtain viscoelastic parameters from our data we use a simple viscoelastic model comprising viscous and elastic elements (i.e., a dashpot and two springs) for the cell body, and an elastic element (a spring) for the cell-substrate adhesion system. Our results show that the spring constants and the viscosity of the cell body components of this viscoelastic model decrease as the temperature increases, whereas the elastic modulus of cell-substrate adhesion increases with temperature. At 37 °C, for the cell body we obtain a value of about 10⁵ P for the viscous element of the viscoelastic model, and a spring constant expressed in units of an elastic modulus of about 10⁴ dyn/cm² for the spring in series with the viscous element, with another spring with a modulus of about 2×10³ dyn/cm² in parallel with these. In comparable units, we have a modulus for the cell-substrate adhesion system of about 3×10³ dyn/cm². Received: 23 March 1998 / Revised version: 23 June 1998 / Accepted: 1 July 1998     

The lateral separation of contacts on erythrocytes agglutinated by polylysine

The form of contact seam (whether a continuous parallel seam or membranes in spatially periodic contact) has been characterized for normal and for neuraminidase pretreated human erythrocytes following adhesion in solutions of polylysine in the molecular mass range 10–225 kDa at concentrations from 0.5 to 1.0 mg/mL. The adhesion contact seam was spatially periodic for all normal control cells in polylysine. The lateral separation of contacts decreased from 1.6 to 0.8 μm as the concentration of 225 kDa polylysine was increased threefold from the adhesion threshold value. The separation distance did not change further even at high polymer concentrations that increased the electrophoretic velocity to positive values over twice the modulus of the velocity of control cells. The probability of cell adhesion decreased at these high polymer concentrations. The lateral contact separation increased and cell adhesion decreased for cells pretreated with neuraminidase. Cell adhesion did not occur when neuraminidase reduced the cell electrophoretic velocity modulus by 30%. Following neuraminidase pretreatments that allowed a small amount of adhesion, the cell contact seam was continuous rather than spatially peridic. The results show that a procedure that increases (e.g., polymer concentration increase) or decreases (e.g., enzyme removal of polycation crosslinking site) attraction leads to shorter (to a limiting value) or longer lateral contact separation, respectively.     

Short-term binding of fibroblasts to fibronectin: optical tweezers experiments and probabilistic analysis

The biophysical properties of the interaction between fibronectin and its membrane receptor were inferred from adhesion tests on living cells. Individual fibroblasts were maintained on fibronectin-coated glass for short time periods (1–16 s) using optical tweezers. After contact, the trap was removed quickly, leading to either adhesion or detachment of the fibroblast. Through a stochastic analysis of bond kinetics, we derived equations of adhesion probability versus time, which fit the experimental data well and were used to compute association and dissociation rates (k ₊=0.3–1.4 s⁻¹ and k _off=0.05–0.25 s⁻¹, respectively). The bond distribution is binomial, with an average bond number ≤10 at these time scales. Increasing the fibronectin density (100–3000 molecules/μm²) raised k ₊ in a diffusion-dependent manner, leaving k _off relatively unchanged. Increasing the temperature (23–37 °C) raised both k ₊ and k _off, allowing calculation of the activation energy of the chemical reaction (around 20 k _B T). Increasing the compressive force on the cell during contact (up to 60 pN) raised k ₊ in a logarithmic manner, probably through an increase in the contact area, whereas k _off was unaffected. Finally, by varying the pulling force to detach the cell, we could distinguish between two adhesive regimes, one corresponding to one bond, the other to at least two bonds. This transition occurred at a force around 20 pN, interpreted as the strength of a single bond. Received: 2 November 1999 / Revised version: 6 March 2000 / Accepted: 19 April 2000     

Atomic force microscopy measurements. Measurements of binding strength between a single pair of molecules in physiological solutions

Atomic force microscopy (AFM) measurements of intermolecular binding strength between a single pair of complementary cell adhesion molecules in physiological solutions provided the first quantitative evidence for their cohesive function. This novel AFM based nanobiotechnology opens a molecular mechanic approach for studying structure to function related properties of any type of individual biological macromolecules. The presented example of Porifera cell adhesion glyconectin proteoglycans showed that homotypic carbohydrate to carbohydrate interactions between two primordial proteogylycans can hold the weight of 1600 cells. Thus, glyconectin type carbohydrates, as the most peripheral cell surface molecules of sponges (today’s simplest living Metazoa), are proposed to the primary cell adhesive molecules essential for the evolution of the multicellularity.     

The strength of integrin binding between neutrophils and endothelial cells

The firm adhesion of activated polymorphonuclear neutrophils to endothelial cells in blood vessels is achieved through binding of the integrin intercellular adhesion molecule. To contribute to the better understanding of this adhesion step, our investigation is aimed at the relationship between integrin expression and the strength of neutrophil binding to endothelial cells. Flow cytometry and 3D scanning microscopy are used to study integrin expression and distribution, respectively. It is found that CD11b/CD18 integrin expression is localized in clusters distributed irregularly over the neutrophil surface. After cell activation, the cluster distribution polarizes, increasing the local CD11b/CD18 density concurrently with nearly doubled integrin expression. The neutrophil adhesion efficiency is measured in a flow chamber coated successively by various substrates, including endothelial cells in an activated state. Analysis of the flow dependence of the number of attached cells reveals the prevailing number of neutrophils with stronger binding to the endothelium when both cells are in the activated state in comparison with non-activated cells.     

Probing elasticity and adhesion of live cells by atomic force microscopy indentation

Atomic force microscopy (AFM) indentation has become an important technique for quantifying the mechanical properties of live cells at nanoscale. However, determination of cell elasticity modulus from the force–displacement curves measured in the AFM indentations is not a trivial task. The present work shows that these force–displacement curves are affected by indenter-cell adhesion force, while the use of an appropriate indentation model may provide information on the cell elasticity and the work of adhesion of the cell membrane to the surface of the AFM probes. A recently proposed indentation model (Sirghi, Rossi in Appl Phys Lett 89:243118, 2006), which accounts for the effect of the adhesion force in nanoscale indentation, is applied to the AFM indentation experiments performed on live cells with pyramidal indenters. The model considers that the indentation force equilibrates the elastic force of the cell cytoskeleton and the adhesion force of the cell membrane. It is assumed that the indenter-cell contact area and the adhesion force decrease continuously during the unloading part of the indentation (peeling model). Force–displacement curves measured in indentation experiments performed with silicon nitride AFM probes with pyramidal tips on live cells (mouse fibroblast Balb/c3T3 clone A31-1-1) in physiological medium at 37°C agree well with the theoretical prediction and are used to determine the cell elasticity modulus and indenter-cell work of adhesion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Intercellular separation forces generated by intracellular pressure

Turgor pressure tends to force plant cells towards a spherical form, thus separating them at the angles from adjacent cells. In cooked vegetables containing starch, the swelling pressure of starch gelatinization generates analogous cell separation forces. A theoretical analysis of the relationship between internal pressure and cell separation forces is presented. Apart from the effect of internal pressure, cell separation forces increase with the diameter of the cell and decrease with the number of cell sides. Cell separation forces are reduced by the introduction of intercellular spaces and decrease further as these expand. The relationship between intracellular pressure and cell separation forces provides a basis upon which the strength of intercellular adhesion can be measured by experiment.     

Cell-cell and cell-substrate adhesion in cultured Drosophila imaginal disc cells

Summary Drosophila imaginal disc cell lines were used to investigate various aspects of cellular adhesion in vitro. The distribution of PS integrins and their involvement in cell-cell and cell-substrate adhesion were assessed with the monoclonal antibody aBG-1 against the βPS subunit, and both forms of adhesion were found to be impeded by the presence of the antibody. Adhesion to a number of extracellular matrix components was investigated, and the cells were found to adhere to human fibronectin. This adhesion was inhibited by aBG-1. The adhesion molecule fasciclin III was also found in these cells. Given that the cells are competent to perform cell-cell and cell-substrate adhesion, it was thought that apical basal polarity might be restored when other suitable conditions were provided, i.e., an artificial basement layer with feeder cells to provide nutrients basally to the cells, and some features of apical-basal morphology were seen in cells cultured under these conditions.     

Comparative analysis of vascular endothelial cell activation by TNF-α and LPS in humans and baboons

As an Old World nonhuman primate, baboons have been extensively used for research on dyslipidemia and atherogenesis. With increasing knowledge about the endothelium's role in the initiation and progression of atherosclerosis, the value of the baboon model can be increased by developing it for research on the role of dysfunctional endothelium in atherogenesis. Toward that goal, we have established and validated methods of isolating and culturing baboon femoral artery endothelial cells (BFAECs) and compared baboon endothelial cellular characteristics with those of humans. Our results indicated that baboon and human endothelial cells share similar growth and culture behaviors. As was the case for human endothelial cells, BFAECs responded to tumor necrosis factor (TNF)-α stimulation with increased expression of adhesion molecules (maximum increase for intracellular adhesion molecule (ICAM): 1.76±0.26-fold; vascular cell adhesion molecule (VCAM): 1.65±0.25-fold; E-selectin: 2.86±0.57-fold). However, BFAECs were hyporesponsive to lipopolysaccharide (LPS) (range, 0.25–20 μg/mL) in adhesion molecule expression, whereas 1 μg/mL LPS induced 2.14- to 3.71-fold increases in human endothelial cells. The differential responses to LPS were not related to TLR-2 and toll-like receptor (TLR)-4 expression on the cell surface. And baboon microvascular endothelial cells had similar features as BFAECs. We observed constitutive expression of interleukin (IL)-6, IL-8, granulocyte macrophage colony-stimulating factor (GM-CSF), and monocyte chemoattractant protein (MCP)-1 in both human and baboon endothelial cells, and these cytokines were further induced by TNF-α and LPS. We also demonstrated that the responses to TNF-α or LPS varied among baboons maintained under the same dietary and environmental conditions, suggesting that response may be controlled by genetic factors.     

Phase transitions of the coupled membrane-cytoskeleton modify cellular shape

Formation of protrusions and protein segregation on the membrane is of a great importance for the functioning of the living cell. This is most evident in recent experiments that show the effects of the mechanical properties of the surrounding substrate on cell morphology. We propose a mechanism for the formation of membrane protrusions and protein phase separation, which may lay behind this effect. In our model, the fluid cell membrane has a mobile but constant population of proteins with a convex spontaneous curvature. Our basic assumption is that these membrane proteins represent small adhesion complexes, and also include proteins that activate actin polymerization. Such a continuum model couples the membrane and protein dynamics, including cell-substrate adhesion and protrusive actin force. Linear stability analysis shows that sufficiently strong adhesion energy and actin polymerization force can bring about phase separation of the membrane protein and the appearance of protrusions. Specifically, this occurs when the spontaneous curvature and aggregation potential alone (passive system) do not cause phase separation. Finite-size patterns may appear in the regime where the spontaneous curvature energy is a strong factor. Different instability characteristics are calculated for the various regimes, and are compared to various types of observed protrusions and phase separations, both in living cells and in artificial model systems. A number of testable predictions are proposed.     

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).     

Biomechanics of cell rolling: shear flow, cell-surface adhesion, and cell deformability

The mechanics of leukocyte (white blood cell; WBC) deformation and adhesion to endothelial cells (EC) has been investigated using a novel in vitro side-view flow assay. HL-60 cell rolling adhesion to surface-immobilized P-selectin was used to model the WBC-EC adhesion process. Changes in flow shear stress, cell deformability, or substrate ligand strength resulted in significant changes in the characteristic adhesion binding time, cell-surface contact and cell rolling velocity. A 2-D model indicated that cell-substrate contact area under a high wall shear stress (20 dyn/cm2) could be nearly twice of that under a low stress (0.5 dyn/cm2) due to shear flow-induced cell deformation. An increase in contact area resulted in more energy dissipation to both adhesion bonds and viscous cytoplasm, whereas the fluid energy that inputs to a cell decreased due to a flattened cell shape. The model also predicted a plateau of WBC rolling velocity as flow shear stresses further increased. Both experimental and computational studies have described how WBC deformation influences the WBC-EC adhesion process in shear flow.     

Microplates: a new tool for manipulation and mechanical perturbation of individual cells.

We present a new type of microinstrument allowing manipulation and mechanical perturbation of individual cells under an optical microscope. These instruments, which we call microplates, are pulled from rectangular glass bars. They have flat tips, typically 2 microm thick x 20 microm wide, whose specific shape and stiffness can be adjusted through the pulling protocol. After appropriate chemical treatment, microplates can support cell adhesion and/or spreading. Rigid microplates are used to hold cells, whereas more flexible ones serve as stress sensors, i.e. their deflexion is used to probe forces in the range of 1-1000 nN. The main advantages of microplates are their simple geometry and surface properties, and their ability to provide mechanical measurements. In this methodological paper, we give details about microplate preparation and adhesiveness, manipulation set-up, force calibration, and image analysis. Several manipulations have already been carried out on fibroblasts, including uniaxial deformation, micropipet aspiration of adherent cells, and cell-substrate separation. Our results to date provide new insights into the morphology, mechanical properties, and adhesive resistance of cells. Many future applications can be envisaged.     

Local calcium transients contribute to disappearance of pFAK, focal complex removal and deadhesion of neuronal growth cones and fibroblasts

Cell adhesion is crucial for migration of cells during development, and cell-substrate adhesion of motile cells is accomplished through the formation and removal of focal complexes that are sites of cell-substrate contact. Because Ca2+ signaling regulates the rate of axon outgrowth and growth cone turning, we investigated the potential role of Ca2+ in focal complex dynamics. We describe a novel class of localized, spontaneous transient elevations of cytosolic Ca2+ observed both in Xenopus neuronal growth cones and fibroblasts that are 2-6 mum in spatial extent and 2-4 s in duration. They are distributed throughout growth cone lamellipodia and at the periphery of fibroblast pseudopodia, which are regions of high motility. In both cell types, these Ca2+ transients lead to disappearance of phosphorylated focal adhesion kinase (pFAK) and deadhesion from the substrate as assessed by confocal and internal reflection microscopy, respectively. The loss of pFAK is inhibited by cyclosporin A, suggesting that these Ca2+ transients exert their effects via calcineurin. These results identify an intrinsic mechanism for local cell detachment that may be modulated by agents that regulate motility.     

Vinculin and Cell-Cell Adhesion

Vinculin, a 117-kDa protein, is a constituent of adhesion plaques and adherence junctions in non-muscle cells. We investigated the role of vinculin on the physical strength of cell-cell adhesion by conducting disaggregation assays on aggregates of parental wild-type F9 mouse embryonal carcinoma cells (clone BIM), two vinculin-depleted F9 cell lines, γ227 and γ229, and a reconstituted γ229 cell line (R3) that re-express vinculin. Immunoblotting demonstrated that the four cell lines used in the study had similar expressions of the cell-cell adhesion molecule E-cadherin and associated membrane proteins α- and β-catenin. Double immunofluorescence analysis showed that, in contrast to the vinculin-null cell lines, BIM and R3 cells expressed abundant vinculin at the cell margins in adhesion plaques and in cell-cell margins that also contained actin. Laminar flow assays showed that both the vinculin-positive and vinculinnegative cell aggregates that were formed in culture in the course of 24 to 48 hours largely remained intact despite the imposition of shear flow at high shear rates. Since laminar flow imposed on cell aggregates act to separate cells from each other, our data indicate that F9 cells that were adherent to a substrate formed strong cell-cell adhesion bonds independent of vinculin expression. On the other hand, aggregates of vinculin-depleted γ229 and γ227 cells that were formed in suspension during a two-hour static incubation at 37°C were desegregated more easily with the imposition of shear flow than the BIM and R3 cell aggregates formed under identical conditions. Loss of vinculin was associated with a reduction in cell-cell adhesion strength only among those cells lacking contact to a substrate. Overall, the results indicate that vinculin is not needed for forming strong cell-cell adhesion bonds between neighboring carcinoma cells which are adherent to the basal lamina.     

Comparison of adhesive bond strength of different cell types in vitro

Summary An established in vitro assay for quantitating cell-substratum adhesion has been utilized to measure the adhesiveness of 10 cell lines to a colloidal overlay. The procedure, a derivation of the William’s blister test for adhesives, involves growing cells to confluency on a polystyrene surface and then overlaying the monolayers with a Bacto-agar substratum. The cell-agar substratum systems are debonded and the^ra,adhesive bond strength, of the separation of the two interfaces calculated. The^ra’s were determined for the following cell types: SGL (gingival epithelial-like), L (transformed mouse fibroblasts), HeLa (human carcinoma), MDCK (canine kidney epithelial), WI-38 (human embryonic lung), Flow 1000 (human embryonic skin—muscle), Flow 4000 (human embryonic kidney), Flow 5000 (whole human embryo), BALB/c 3T3 (mouse fibroblasts) and SV₄₀-transformed BALB/c 3T3 (simian virus 40-transformed mouse fibroblasts). Transformed cells (L, HeLa and SV₄₀-transformed BALB/c 3T3) proved to be quantitatively less adhesive (^ra/cell) than either fibroblast or epithelial-like cell lines. Of the “normal” cells tested the kidney cells, human embryonic fibroblast and canine epithelial cells, and the gingival epithelial-like cells demonstrated a weaker binding to the colloidal overlay than the mouse fibroblasts (BALB/c 3T3), the human embryonic lung, the human embryonic skin-muscle, and the whole human embryo fibroblast cell lines. Concanavalin A increased the bonding strength of Flow 5000 cells after 24 hr incubation; however, the adhesiveness of the treated BALB/c 3T3 cells remained similar to the unterested samples while the^ra of the treated SV₄₀-transformed BALB/c 3T3 cells decreased. This research was supported by National Institute of Dental Research Grant DE03983.