Isolation of centrolobular and perilobular hepatocytes after phenobarbital treatment

Daily phenobarbital (PB) injections, on 3-7 consecutive days, induce an intense proliferation of smooth endoplasmic reticulum (ER) associated with a decrease of the glucose-6-phosphatase activity. This situation first affects the centrolobular hepatocytes, enhancing the degree of liver lobule heterogeneity. This experimental model was used for isolation and further subfractionation of hepatocytes on Ficoll density gradients, as described in the preceding paper. Profiles of protein, DNA, RNA, glycogen, phosphorylase, and glucose-6-phosphatase were determined all along the gradient. Two liver cell populations were distinguished: (a) light hepatocytes (mean density 1.10) present the same morphological characteristics as centrolobular cells, i.e., an abundant smooth ER composed of tubular elements, numerous small mitochondria, and few glycogen particles; (b) heavy hepatocytes (mean density 1.14) are characterized by large and compact glycogen areas and prominent rough endoplasmic cisternae, as are the perilobular cells. After incubation in the Wachstein-Meisel medium, Centrolobular hepatocytes exhibit dispersed reaction sites of glucose-6-phosphatase activity, whereas perilobular cells present a continuous and intense reaction. Morphometric determinations were carried out for both cell populations. Centrolobular PB hepatocytes are considerably enlarged (mean diameter: 23.7 mum); perilobular hepatocytes have a significantly smaller mean diameter of 19.2 mum, which is close to the values of control liver cells.     

Isolation of inclusion bodies from rabbit lung parenchyma

The mitochondrial-plus-lysosomal fraction of rabbit lung parenchyma was studied by equilibrium density centrifugation in continuous sucrose density gradients (specific gravity 1.035 to 1.250). High concentrations of lysosomal marker enzymes were found both in a broad band at density 1.15–1.18, a density typical for lysosomes, and in a band at density 1.06–1.07. This light density band also had the highest specific activity of phospholipid, which thin layer and gas-liquid chromatography showed to be primarily lecithin with a high content of palmitic acid residues. Electron microscopy of material from the light density band showed a homogeneous array of particles which bear a strong resemblance to the inclusion bodies of the type II alveolar epithelial cell as seen in electron micrographs of rabbit lung tissue sections. These data suggest that the light density band is an isolation of intact type II alveolar epithelial cell inclusion bodies, which previous studies have implicated as the storage site of the phospholipid moiety of pulmonary surfactant.     

The density of apical cells of dark-grown protonemata of the mossCeratodon purpureus

Summary Determinations of plant or algal cell density (cell mass divided by volume) have rarely accounted for the extracellular matrix or shrinkage during isolation. Three techniques were used to indirectly estimate the density of intact apical cells from protonemata of the mossCeratodon purpureus. First, the volume fraction of each cell component was determined by stereology, and published values for component density were used to extrapolate to the entire cell. Second, protonemal tips were immersed in bovine serum albumin solutions of different densities, and then the equilibrium density was corrected for the mass of the cell wall. Third, apical cell protoplasts were centrifuged in low-osmolarity gradients, and values were corrected for shrinkage during protoplast isolation. Values from centrifugation (1.004 to 1.015 g/cm³) were considerably lower than from other methods (1.046 to 1.085 g/cm³). This work appears to provide the first corrected estimates of the density of any plant cell. It also documents a method for the isolation of protoplasts specifically from apical cells of protonemal filaments.Abbreviations BSA bovine serum albumin - ER endoplasmic reticulum - V_v volume fraction     

Isolation of motile spermatozoa by density gradient centrifugation in Percoll®

A procedure using centrifugation in density gradients composed of Percoll was developed for isolation of spermatozoa from mammalian semen. To evaluate the technique, rabbit, human, or bovine semen was layered over continuous Percoll gradients ranging in density from 1.02 to 1.13 gm/ml and centrifuged at 1,500g for 45 min. After centrifugation, the seminal plasma remained above the gradient, whereas the spermatozoa and seminal particles were distributed within the gradient according to their buoyant densities. Unlike most washing techniques, no sperm pellet was formed; instead, the spermatozoa were concentrated into a compact band above the most dense layer of Percoll. The spermatozoa recovered from the gradient were easily resuspended by gentle techniques. Thus, the mechanical stress to the spermatozoa was minimized. Osmotic stress to the spermatozoa was also negligible as the Percoll gradients were isotonic throughout. Spermatozoa obtained by this technique possessed motility equivalent to that of spermatozoa in the unfractionated semen. Sperm suspensions recovered from the gradients contained less than 5% of the nonspermatozoal particles present in the original samples of unfractionated semen. Soluble seminal components were also efficiently removed from the spermatozoa. Thirty billion bovine spermatozoa could be fractionated on a single gradient without loss of effectiveness. Recovery of spermatozoa from these preparative separations averaged 80%. These results demonstrated that Percoll was a superior medium for efficient density gradient isolation of motile spermatozoa free of contamination by other seminal constituents.     

Separation of Dictyostelium discoideum cells into density classes throughout their development and their relationship to the later cell types

This paper describes a fast, non-destructive method for the separation of large quantities of Dictyostelium discoideum cells into density classes at all stages of development. The cells were separated by low-speed centrifugation on preformed, linear Percoll density gradients. On these gradients, cells at all developmental stages showed a unimodal variation in density and this variation in density rapidly increased during the first hours of development. The density was affected by the amount of salt present in the gradient medium, which suggests that it is regulated by a permeability property of the cells. Slug cells showed a unimodal variation in density and did not form bands corresponding to the cell types. However, were able to isolate density fractions which showed a good enrichment of prespore and prestalk cells: 95% and 90%, respectively. Preaggregation cells separated on density gradients yielded fractions which contained different amounts of three developmentally regulated enzymes. Hence, cells at this stage are already heterogeneous in their enzymatic content. Sorting experiments showed a strong correlation between density and developmental fate; the least dense (light) cells preferentially became prestalk cells, and the dense (heavy) cells became prespore cells. This was found for cells at all developmental stages; even vegetative-stage cells showed considerable heterogeneity with regard to density, which was related to their developmental fate. The light cells become prestalk cells, and the heavy cells become prespore cells. Vegetative cells from the various density fractions differed in their DNA content and temporal onset of mitotic activity when resuspended in medium. Therefore, we suggest that the separation of vegetative cells on density gradients results in a separation of cells into cell-cycle phases. Hence, there appear to be cell-cycle-linked differences among vegetative cells, which bias their differentiation towards either the spore or stalk pathway.     

Isolation of separate apical,lateral and basal plasma membrane from cells of an insect epithelium. A procedure based on tissue organization and ultrastructure

The tissue used in this study was the midgut of the tobacco hornworm larva, Manduca sexta. The midgut epithelium is a single layer of cells resting on a thin basal lamina and underlying discontinuous muscle layer. The epithelial cells are of two main types, goblet and columnar cells, joined together by the septate junctions characteristic of insect epithelia. From this tissue we were able to isolate four distinct plasma membrane fractions; the lateral membranes, the columnar cell apical membrane, the goblet cell apical membrane and a preparation of basal membranes from both cell types. The lateral membranes were isolated by density gradient centrifugation following gentle homogenization of the midgut hypotonic medium, which caused the cells to rupture at their apical and basal surfaces, releasing long segments of lateral membranes still joined by their septate junctions. For isolation of apical and basal membranes the tissue was disrupted by ultrasound, based on the light microscopic observation that carefully controlled ultrasound can be used to disrupt each cell in layers starting at the apical surface. The top layer contained the columnar cell apical membrane, which consists of microvilli forming a brush border covering the lumenal surface of the epithelium. The second layer contained the goblet cell apical membrane, which is invaginated to form a cavity occupying the apical half of the cell, and the third layer contained the basal membranes. As each layer was stripped off the epithelium it was collected and the plasma membrane purified by differential or density gradient centrifugation. For all four membrane fractions, the isolation procedure was designed to preserve the original structure of the membrane as far as possible. This allowed electron microscopy to be used to follow each step in the isolation procedure, and to identify the constituents of each subcellular preparation. Although developed specifically for M. sexta midgut, these techniques could readily be modified for use on other epithelia.     

Fetal erythroblast isolation up to purity from cord blood and their culture in vitro

BACKGROUND: Erythroblasts have been the most encouraging candidate cell type for noninvasive prenatal genetic investigation. We previously showed that human erythroblasts can be recovered from bone marrow and blood bank buffy coats by a physical cell separation. In the present study, we modified our previous methodology, taking into account the peculiar behavior of erythroblasts in response to modifications of pH and osmolality of the separation medium. METHODS: Twenty to forty milliters of cord blood were initially centrifuged on Ficoll/diatrizoate (1.085 g/ml). The interphase cells were further separated on a continuous density gradient (1.040-1.085 g/ml). Two different gradients were initially compared: the first was iso-osmolar and neutral, whereas the second also contained an ionic strength gradient and a pH gradient (triple gradient). A subsequent monocyte depletion was performed by using magnetic microbeads coated with anti-CD14 monoclonal antibody (mAb), and erythroblasts were purified by sedimentation velocity. Purified cells were investigated by analyses with fluorescence-activated cell sorting (FACS) and fluorescence in situ hybridization (FISH) and immunocytochemistry with mAb against fetal hemoglobin and were cultured in vitro. RESULTS: When nucleated cells were spun on an iso-osmolar and neutral continuous density gradient, two separated bands of nucleated red blood cells (NRBCs) were obtained: a light fraction banding at 1.062 g/ml and an heavy fraction banding at 1.078 g/ml. Conversely, when cells were spun in the triple gradient, NRBCs were shifted to the low-density region. Monocyte depletion by immunomagnetic microbeads and velocity sedimentation provided a pure erythroblast population. FACS and FISH analyses and immunocytochemistry substantiated the purity of the isolated cell fraction, which was successfully cultured in vitro. CONCLUSIONS: We have shown that fetal erythroblasts can be purified up to homogeneity from cord blood, but further refinements of the isolation procedure are necessary before the same results can be obtained from maternal peripheral blood.     

Functional and physical characteristics of rat Leydig cell populations isolated by metrizamide and Percoll gradient centrifugation

The physical and functional properties of Leydig cell populations obtained by centrifugation of testicular cells in two different density gradient media, Percoll and Metrizamide, were compared. Percoll-gradient centrifugation yielded two Leydig cell bands (Peak I and Peak II) that were comparable, as to their density and testosterone-producing capacity, to the respective Leydig cell bands, Population I and Population II, isolated in a Metrizamide gradient. The denser Leydig cell band (II) had a greater capacity for testosterone production than the less dense band (I), regardless of the type of gradient used for its isolation. Metrizamide gradient centrifugation separated the majority of germ cells from the "light" (Population I) Leydig cells, whereas in the Percoll gradient, germ cells comigrated with Peak I Leydig cells. Leydig cell separation by Percoll gradients was highly dependent on the presence of Ca2+ and Mg2+ in the medium, while these cations had no effect on the separation of Leydig cells by Metrizamide. In conclusion, Metrizamide gradient centrifugation yielded two Leydig cell populations of similar functional and physical properties to the respective populations isolated in Percoll gradients.     

Stathmokinetic Analysis of Human Epidermal Cells in vitro

Proliferation kinetics of cultured human epidermal cells is characterized in quantitative terms. Three distinct subpopulations of keratinocytes, two of which are cycling have been discriminated by two parameter DNA/RNA flow cytometry. Based on mathematical modelling, the cell cycle parameters of the cycling subpopulations have been assessed from stathmokinetic data collected at different time points after initiation of cultures (7–15 days). the first subpopulation is composed of low-RNA cells which resemble basal keratinocytes of epidermis and which show some characteristics of stem cells; these cells have a mean generation time of approximately 100 hr. the second subpopulation consists of high-RNA cells, resembling stratum spinosum cells of epidermis, which have an average generation time of approximately 40 hr. the third subpopulation consists of non-cycling cells with G_o/G₁ DNA content, with cytochemical features similar to those of cells in granular layer of epidermis. The results based on modelling can reproduce with acceptable accuracy the actual growth curve of the cultured cell population. Analysis of kinetics and differentiation of human keratinocytes is of interest in view of the recent application of cultured epidermal cell sheets for transplantation onto burn wounds. the results of this study also reveal the existence of regulatory mechanisms associated with proliferation and differentiation in the cultured epidermal cell population.     

Stathmokinetic analysis of human epidermal cells in vitro

Proliferation kinetics of cultured human epidermal cells is characterized in quantitative terms. Three distinct subpopulations of keratinocytes, two of which are cycling, have been discriminated by two parameter DNA/RNA flow cytometry. Based on mathematical modelling, the cell cycle parameters of the cycling subpopulations have been assessed from stathmokinetic data collected at different time points after initiation of cultures (7-15 days). The first subpopulation is composed of low-RNA cells which resemble basal keratinocytes of epidermis and which show some characteristics of stem cells; these cells have a mean generation time of approximately 100 hr. The second subpopulation consists of high-RNA cells, resembling stratum spinosum cells of epidermis, which have an average generation time of approximately 40 hr. The third subpopulation consists of non-cycling cells with G0/G1 DNA content, with cytochemical features similar to those of cells in granular layer of epidermis. The results based on modelling can reproduce with acceptable accuracy the actual growth curve of the cultured cell population. Analysis of kinetics and differentiation of human keratinocytes is of interest in view of the recent application of cultured epidermal cell sheets for transplantation onto burn wounds. The results of this study also reveal the existence of regulatory mechanisms associated with proliferation and differentiation in the cultured epidermal cell population.     

Analysis of human cytomegalovirus nucleoprotein complexes

When chromatin was isolated from cells infected with human cytomegalovirus, the virus DNA remained with the chromatin fraction. If deproteinized virus DNA was added to either isolated nuclei or chromatin, the DNA was lost during the chromatin isolation. When isolated chromatin from cytomegalovirus-infected cells was banded in isopycnic metrizamide gradients, a single peak with a density of 1.18 g/cm3 was present. Analysis of this peak in isopycnic neutral CsCl gradients indicated that it contained both human cytomegalovirus and human embryonic lung cell DNAs. When infected nuclei were treated with micrococcal nuclease, 11S subunit particles which cosedimented with cell nucleosomes and contained virus DNA were isolated.     

Golgi studies of the first optic ganglion of the ant,Cataglyphis bicolor

The neurons of the first optic ganglion (the lamina) in the desert ant, Cataglyphis bicolor, have been studied with the light microscope after Golgi silver impregnation. The different types of retinal and laminal fibres and their configuration are compared with the results obtained in the bee. The first synaptic region in the visual system of the ant lies proximally to the fenestrated layer below the basement membrane and the layer containing the monopolar cell bodies. The synaptic region can be separated into three morphologically different zones: (1) The most distal layer where the short visual fibres end at two different levels. The short visual fibres and some laminal fibres (monopolar cell fibres) also show lateral elements in this region. (2) The second layer appears almost free of branches of retinal and laminal fibres. (3) The most proximal layer, which has a characteristically dense horizontal structure resulting from the lateral elements of long visual, centrifugal, monopolar and tangential fibres. Nine cell axons arising from each ommatidium leave the retina. Six of these are short visual fibres and end at two different levels in the lamina. Three different types of short visual fibres can be distinguished by their different terminal depths and lateral branching pattern. The remaining three fibres, the long visual fibres, terminate in the medulla. They can be distinguished from each other by their lateral elements in the lamina neuropile. The five morphologically different laminal fibre types (axons of the monopolar cells in the lamina) have different shapes and different arborizations at different levels. Tangential, centrifugal and incerta sedis-fibres, which originate either from cell bodies in the cell body layer at the periphery of the outer chiasma or more centrally, terminate in the synaptic region of the lamina. Consideration is given to the clearly demarkated arrangement and length of the branching pattern of retinal and laminal fibres at different levels of the synaptic region of the lamina. In addition, a hypothetical connectivity pattern is discussed.     

The Differentiation of Contact Cells and Isolation Cells in the Xylem Ray Parenchyma of Populus maximowiczii

A histochemical analysis was made of the differentiation ofcontact cells and isolation cells in the xylem ray parenchymaof Populus maximowiczii. The contact cells formed secondarywalls at approximately the same time as adjoining vessel elements.The lignification of the cell walls of contact cells and vesselelements began earlier than that of wood fibres and isolationcells. Thus, the formation of the secondary wall, includinglignification, of the contact cells might occur at the sametime as that of the vessel elements to which they are directlyconnected. By contrast, the isolation cells began to form secondarywalls later than the vessel elements and wood fibres in thevicinity of the isolation cells. After the deposition of thesecondary wall, a protective layer was formed in contact cellsbut no isotropic layer was observed in isolation cells. Theresults suggest the importance of vessel elements in the determinationof the differentiation of adjoining ray parenchyma cells.Copyright1999 Annals of Botany Company Contact cell, isolation cell, vessel element, xylem differentiation, Populus maximowiczii Henry.     

Isolation and culture of somatotrophs from the pituitary of the rainbow trout: Immunological and physiological characterization

Summary The aim of the present paper was to obtain somatotroph- and gonadotroph-enriched populations from collagenase dispersed pituitaries of male rainbow trout. Inasmuch as the percentage of immunoreactive gonadotrophs and somatotrophs present in pituitaries was higher at spermiation than at the beginning of spermatogenesis, we tried such a cell separation with fish at this stage of spermatogenesis. Cells were fractionated using their differences in buoyant density with centrifugation in Percoll solutions. The use of Percoll linear gradients (1.110 to 1.027 g/ml) showed that somatotroph cells have a density of between 1.102 and 1.064 g/ml whereas gonadotrophs are spread over the range of the gradient. It was thus possible, by using linear or discontinuous Percoll gradients, to obtain 95 to 67% (mean 80%) enriched somatotropic cell fractions while no enriched gonadotropic cell fractions were collected. The fractionated cells kept their ability to be cultured and to be responsive to specific secretagogues. Somatostatine induced a 80 to 85% decrease in growth hormone release per somatotroph in the initial cell suspension as well as in the different cell fractions. On the other hand, the basal growth hormone release per cell was lower in the fractions containing cells with a density lower than 1.062 g/ml. Inversely, the gonadotrophs have a basal release per cell independent of their density, and this is also available for their responsiveness to salmon gonadotropin-releasing hormone.     

Isolation and differentation of cloned epithelial cell lines from normal rat mammary glands

Summary Single-cell-cloned cell lines have been established from primary cultures of neonatal rat mammary glands. A representative cuboidal cell line, Rama 704, shows the presence of intermediate filamental proteins keratin and vimentin, and occasional cells express milk fat globule membrane antigens on their apical surfaces. Rama 704 cells grow as a cuboidal pavement in culture and produce hemispherical blisters or domes when confluent. Noteworthy ultrastructural features are the presence of junctional complexes, desmosomes, and apical microvilli typical of epithelia. Cells seeded within floating collagen gels with form a variety of multicellular outgrowths, some of which are ductlike in morphology and are composed of polarized cells surrounding a central lumen. The cuboidal cells produce elongated cells under conditions of high cell density and also when cells float off collagen gels and reattach to the plastic substrate. The former elongated cells have been cloned and three cel lines established: Rama 710, 711, and 712; the latter uncloned elongated cells are termed Rama 704E. The cloned elongated cells show an increase in the amounts of basement membrane proteins deposited, a lack of junctional complexes and microvilli, and an increase in the amount of rough endoplasmic reticulum compared with their parental cells. Rama 704E cells show an enhanced deposition of basement membrane proteins and increased amounts of actin in the cytoplasm over the elongated cell lines and contain microfilaments and pincocytotic vesicles similar to those seen in myoepithelial cells. All the elongated cells and lines fail to form ductlike structures within collagen gels. None of the cell lines form tumors in syngeneic rats although they all produce some tumors in nude mice, which are composed of cords of epithelioid cells and spindle cells in varying proportions. In addition, some of the Rama 704 tumors contain rhabdomyoblastic elements that penetrate the host fat pad. This is the first report of the isolation and characterization of a stable cuboidal cell line from a neonatal rat mammary gland. The Rama 704 cell line shows morphological and biochemical features of mammary epithelial cells and converts at high cell density to elongated cells that have also been cloned.     

Conditions for antitumor cytotoxic T-lymphocyte formation and inhibition of their activity by T-suppressors in mixed culture of human lymphocytes and tumor cells

Conditions for antitumour autolytic T lymphocytes (CTL) induction during human mixed lymphocyte tumour cell culturing (MLTC), as well as the patterns of CTL activation abolition in the experimental system by suppressor cells were investigated. The responders used in MLTC were peripheral blood lymphocytes of colorectal cancer patients fractionated by means of multilayer Percoll gradients centrifugation (the density of layers being 1.077, 1.067, 1.056 g/ml). The cells of the second fraction collected in the density interphase of 1.077-1.067 g/ml (more than 90% of population belonging to T lymphocytes), when used as responders in MLTC, developed an autolytic activity against autologous and allogeneic tumour target cells. The cell of the first fraction (collected in the interphase of 1.067-1.056 g/ml) added to the second fraction cells at the beginning of MLTC, prevented the following CTL induction. The first fraction contained T-suppressor cells capable of strongly interfering with antitumour CTL activity.     

Assessment of different protocols for the isolation and purification of gut associated lymphoid cells from the gilthead seabream (<Emphasis Type="Italic">Sparus aurata</Emphasis> L.)

Teleost gut associated lymphoid tissue (GALT) consists of leucocyte populations located both intraepithelially and in the lamina propria with no structural organization. The present study aims to assess different protocols for the isolation of GALT cells from an important fish species in the Mediterranean aquaculture, the gilthead seabream. Mechanical, chemical and enzymatic treatments were assayed. Nylon wool columns and continuous density gradients were used for further separation of cell subpopulations. Light microscopy and flow cytometry showed that the highest density band (HD) consisted of a homogeneous lymphocytic population, whereas the intermediate density band (ID) corresponded to epithelial and secretory cells and some lymphocytes. Respiratory burst activity of total cell suspensions revealed very low numbers of potential phagocytic cells, reflecting results from light microscopy and reports in other teleost species. The present data set up the basis for future functional characterization of GALT in seabream.     

Formation of Interfamilial Cell Symplast During Cell Co-Culture in Vitro

Two cell lines, white tobacco (Nicotiana tabacum) cell line and green carrot (Daucus carota) cell line, each with very distinct cellular structure markers by which the two cell lines could be identified at levels of callus cells, with light microscopy and electron microscopy, were established and used for interfamilial cell co-culture in which callus cells were well mixed and finely dispersed and treated with K+ hypotonic solution. Variegated interfamilial chimeral calli were observed after 10 to 15 days of co-culture. An isolation layer was formed and became thickened at the interface between the two attached unrelated callus cells and the heteroplastic cell wall complex was gradually established. Then the isolation layer became thinned and disappeared and plasmodesmata were formed secondarily within the thinned region and the interfamilial cell symplast was established. The wall in the region with isolation layer was about twice as thick as the fused cell wall of the symplastic. The developmental process of the interfamilial cell symplastic connection was discussed and it was suggested that (1) the nonspecific formation of isolation layer as initial adhesion between the two attached unrelated cells was the prerequisit for symplastic connection de novo formation, and (2) the specific cell recognition leading to disappearance or thickening to lignification or suberization of isolation layer and formation of plasmodesmata started within the isolation layer.     

Human spermatogenic cell marker proteins detected by two-dimensional electrophoresis

Highly homogeneous populations of human pachytene spetmatocytes and round spermatids have been obtained from normal adult testis using unit gravity (STA-PUT) sedimentation. Contaminating Leydig cells have been removed by density centrifugation in discontinuous Percoll gradients to yield resultant germ cell purities of 90–95% for pachytene spermatocytes and 89–96% for round spermatids. The total cellular polypeptide composition of separated human germ cells has been analyzed by two-dimensional polyacrylamide gel electrophoresis to compare 1) human and mouse pachytene spermatocytes (species specificity), 2) samples of human spermatocytes obtained from different individuals (allo specificity), and 3) pachytene spermatocytes and round spermatids from the same patients (stage specificity). Mouse and human germ cells have been found to exhibit extensive homology, but identified marker proteins limited to human spermatocytes include a group of polypeptides at p45/5.9 as well as a protein at p67/5.2. Proteins unique to mouse germ cells include component p65/5.5. Comparisons between different preparations of human pachytene spermatocytes have revealed about 90% electrophoretic homology, but some striking allotypic variations have been noted including the proteins at p45/5.9. Finally, presumptive stage-specific spermatogenic cell markers have been identified including the p45/5.9 polypeptides that are present only in human spermatocytes. Although the physiological roles of particular marker proteins have not yet been determined, the present findings indicate that purified spermatogenic cell populations may be analyzed biochemically to identify constituents important in the regulation of sperm development in man.     

Tumor cell haptotaxis on covalently immobilized linear and exponential gradients of a cell adhesion peptide

The movement of cells up an adhesive substratum gradient has been proposed as a mechanism for directing cell migration during development and metastasis. Critical evaluation of this hypothesis (haptotaxis) benefits from the use of quantifiable, stable substratum gradients of biologically relevant adhesion molecules. We report covalent derivatization of polyacrylamide surfaces with quantifiable gradients of a nonapeptide containing the adhesive Arg-Gly-Asp sequence. Cell migration was studied by seeding derivatized surfaces evenly with B16F10 murine melanoma cells. Within 8 hr, cells on gradients redistributed markedly; higher cell densities were found at gel positions having higher immobilized peptide densities. In contrast, cells seeded on control gels with uniform concentrations of adhesive peptide did not redistribute. Redistribution occurred on gradients in both serum-free and serum-containing media. Experiments with uniform density peptide-derivatized gels demonstrated that redistribution on gradients was not due to preferential initial cell attachment or preferential growth on the higher density of immobilized peptide, but must have been due to cell translocation. Cells on exponential gradients of immobilized peptide migrated to a position on the gel surface corresponding to the highest immobilized peptide density, while cells on linear gradients of the same peptide migrated to a position of intermediate peptide density. These data suggest that the B16F10 cells respond to proportional changes in immobilized peptide density rather than to absolute changes, implying a sensing mechanism which utilizes adaptation. These results demonstrate that (1) a gradient of a small adhesive peptide is sufficient to generate redistribution of cell populations and (2) controlled quantifiable substratum gradients can be produced and used to probe the underlying cellular mechanisms of this behavior.