Continuous Percoll Density Gradient Research Articles

Continuous Percoll Gradient Centrifugation of Erythrocytes—Explanation of Cellular Bands and Compromised Age Separation

(1) Background: When red blood cells are centrifuged in a continuous Percoll-based density gradient, they form discrete bands. While this is a popular approach for red blood cell age separation, the mechanisms involved in banding were unknown. (2) Methods: Percoll centrifugations of red blood cells were performed under various experimental conditions and the resulting distributions analyzed. The age of the red blood cells was measured by determining the protein band 4.1a to 4.1b ratio based on western blots. Red blood cell aggregates, so-called rouleaux, were monitored microscopically. A mathematical model for the centrifugation process was developed. (3) Results: The red blood cell band pattern is reproducible but re-centrifugation of sub-bands reveals a new set of bands. This is caused by red blood cell aggregation. Based on the aggregation, our mathematical model predicts the band formation. Suppression of red blood cell aggregation reduces the band formation. (4) Conclusions: The red blood cell band formation in continuous Percoll density gradients could be explained physically by red blood cell aggregate formation. This aggregate formation distorts the density-based red blood cell age separation. Suppressing aggregation by osmotic swelling has a more severe effect on compromising the RBC age separation to a higher degree.

Purification of Leaf Mitochondria from Arabidopsis thaliana Using Percoll Density Gradients.

The study of plant mitochondria often requires isolation of mitochondria from plant tissues in intact and functional form. Here, we describe an effective procedure of mitochondrial isolation from leaf tissues and whole seedlings of the model dicot species Arabidopsis thaliana by using differential centrifugation and continuous Percoll density gradients.

Subcellular Fractionation of Hela Cells for Lysosome Enrichment Using a Continuous Percoll-Density Gradient.

The enrichment of lysosomes is a useful way to study their structure and function. These dynamic vesicles can be enriched from cell cultures in a variety of ways including immunoprecipitation and fluorescence-activated organelle sorting. These methods are extremely precise but often require the transfection and expression of an affinity or fluorophore-tagged lysosomal membrane protein. A simpler approach uses differential density of subcellular organelles, which are characteristic to a particular type of organelle. Separation of organelles along a density-gradient enables fractionation to enrich for specific organelles (such as lysosomes) in their native state. This protocol outlines an optimized method for enriching lysosomes from HeLa cells with a continuous density-gradient that contains Percoll. Gentle cell lysis and extraction conditions yield dense-fractions that are enriched with functional and intact lysosomes, which can be assayed in downstream analyses. This method is quick (conducted in less than 2 h after harvesting cells), and can be easily scaled and optimized for other cell types.

CENTRIFUGAÇÃO DE ESPERMATOZOIDES OVINOS EM GRADIENTE DE DENSIDADE CONTÍNUO: EFEITO SOBRE A TAXA DE PRENHEZ E PROPORÇÃO DO SEXO

Este trabalho teve como objetivo avaliar a eficiência de gradientes de densidade contínuos de Percoll e OptiPrep para a separação de espermatozoides portadores do cromossomo X, e avaliar a influência sobre a viabilidade espermática, taxa de prenhez e proporção do sexo. Para isso, 100 milhões de espermatozoides foram depositados em cada tubo contendo o gradiente de densidade, previamente preparados, em tubos de poliestireno, 24 horas antes da centrifugação e mantidos a 4 °C. Centrifugou-se a 500 x g por 15 minutos a 22 °C e, posteriormente, os sobrenadantes foram aspirados e os espermatozoides recuperados no fundo dos tubos. Uma amostra foi avaliada antes a após a centrifugação para verificação da motilidade e vigor espermático. Os espermatozoides foram utilizados para a inseminação artificial em tempo fixo. Os resultados indicaram que a centrifugação não afetou a viabilidade espermática e a taxa de prenhez, entretanto não houve desvio da proporção do sexo para fêmeas. Mais estudos são necessários para determinar a necessidade de adaptação da técnica de centrifugação para a espécie ovina e assim obter o desvio da proporção do sexo como na espécie bovina.

Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins

Killer lymphocytes induce apoptosis by the release of cytotoxic mediators from specialized secretory lysosomes, called cytotoxic granules, into the immunological synapse formed with a cell targeted for elimination. Methods are presented here for isolating CTL and NK cell cytotoxic granules using cell disruption by nitrogen cavitation followed by continuous Percoll density gradient fractionation. Protocols are also given for purifying the key cytolytic molecules (perforin, granzyme A, granzyme B, and granulysin) from isolated cytotoxic granules by fast protein liquid chromatography.

SEPARAÇÃO DE ESPERMATOZOIDES PORTADORES DO CROMOSSOMO X BOVINO POR CENTRIFUGAÇÃO EM GRADIENTE DE DENSIDADE CONTÍNUO DE PERCOLL E OPTIPREP: EFEITO SOBRE A VIABILIDADE ESPERMÁTICA E NA PRODUÇÃO IN VITRO DE EMBRIÕES

The aim of this study was to separate X-bearing bovine sperm by continuous Percoll and OptiPrep density gradients and to validate the sexing of resultant in vitro produced embryos by Polimerase Chain Reaction (PCR). Frozen/thawed sperm was layered on density gradients which were previously prepared in polystyrene tubes, 24 h before procedures and maintained at 4 °C. The tubes were centrifuged at 500 x g for 15 min at 22 °C. Supernatants were gently aspirated and the sperm recovered from the bottom of the tubes. Viability and integrity of sperm were evaluated by Trypan Blue/Giemsa stain. Cleavage and blastocyst rates were determined by in vitro production of embryos and PCR was performed for identification of the embryos’ genetic sex. No damage in viability and acrossomal integrity and in cleavage and blastocyst rates was found in the Percoll and OptiPrep treatment compared to the non-centrifuged group (P>0.05). The percentage of female embryos in the Percoll and OptiPrep group was 63.0 and 47.6%, respectively. The female embryos in control group were 48.7%. A sexual deviation in the Percoll density gradient was achieved without reduction of sperm viability and in vitro production rates. KEY WORDS: Bovine, centrifugation, in vitro production of embryos, PCR, X-bearing sperm.

Intrathymic deficient expansion of T cell precursors in Down syndrome

To correlate the intrinsic cellular immunodeficiency, which is a major cause of increased susceptibility to polytopic infections in Down syndrome (DS) patients, with the histologic abnormalities observed in the thymus of these patients, we have studied thymus fragments and thymocyte cell suspensions from 15 non-institutionalized DS subjects. Comparing to the control age-matched samples, a reduced thymic cortex and a distinct depletion of CD1-positive (+) cells was observed by immuno-histologic examination. The phenotypic analysis of unselected thymocytes showed a significant reduction of CD3+, CD1+, CD4+, and CD8+ cells. When the total thymocyte population was separated into 10 fractions, using a continuous Percoll density gradient, a difference in cell distribution was observed. DS thymuses are almost devoid of high-density thymocytes (fractions 6-9) while more than 75% of the cells were recovered in the lightest 3 fractions (Frs). In addition, these thymuses were characterized by a marked depletion of CD1+ cells and by a conspicuous reduction of CD3+ cells normally present in the high-density Frs. On the other hand, the lightest 3 Frs of DS subjects were enriched in low-density CD1+ cells. Although enriched in these cells, normally characterized by a high mitotic activity, Fr1 DS thymocytes showed a reduced spontaneous proliferative capacity. When the expression of T cell receptor alpha- and beta-subunits was studied, the percentages of cells stained with anti-alpha and anti-beta antisera were found to be reduced in DS unfractionated thymocytes. The reduced number of high-density CD1+ thymocytes associated with a reduced spontaneous proliferative activity of low-density CD1+ thymocytes suggests that in DS thymuses there is a deficient expansion of immature T cells, resulting in a reduction of the various thymocyte subpopulations, including the thymocyte pool which differentiates into functionally mature T cells expressing the alpha-beta T cell receptor.

BRYOPSIS MAXIMA (CHLOROPHYTA) GLUTAMATE DEHYDROGENASE: MULTIPLE GENES AND ISOZYMES

Subcellular localization of glutamate dehydrogenase (GDH) was investigated in the green alga Bryopsis maxima. Both intact and pure chloroplasts and mitochondria were isolated by two methods: successive centrifugation and continuous Percoll density gradient centrifugation. The NADP‐dependent GDH activities of the chloroplastic, mitochondrial, and cytosolic portions were estimated as 64.3, 9.8, and 25.9%, respectively, and NAD‐dependent GDH activity was observed only in the chloroplasts. Three organelle‐specific isozymes—chloroplastic NADP‐GDH1, cytosolic/mitochondrial NADP‐GDH2, and cytosolic/mitochondrial NADP‐GDH3—were purified. The molecular masses of these isozymes were estimated to be the same (280 kDa). Km values of NADP‐GDH1, NADP‐GDH2, and NADP‐GDH3 for NADPH in the amination reaction were 30, 110, and 34 μM, respectively, and those for NADH were 185, 1490, and 974 μM, respectively, showing different cofactor affinities. Several NADP‐GDHs and one NAD‐GDH were induced in the chloroplasts during incubation of the collected thalli in either continuous light or darkness in aerated seawater for 0 to 5 days, whereas the cytosolic and mitochondrial NADP‐GDHs decreased to an almost undetectable level in 5 days. Two distinct DNA fragments (BmF‐1 and BmF‐2) encoding B. maxima Okamura GDH were identified and sequenced. They showed 90% homology in their deduced amino acid sequences, whereas synonymous nucleotide substitution was observed in the third position of 52% of the codons. Genomic Southern analysis suggested that the two genes are located at two different loci on the B. maxima chromosome. Thus, B. maxima GDH has been confirmed to be multiple in terms of both protein and gene. The localization of other nitrogen‐assimilating enzymes was also determined. Glutamine synthetase was located in the chloroplasts and the cytosol, glutamate synthase was located in the chloroplasts, and nitrate reductase was located in the cytosol.

Isolation of bovine neutrophils with biomagnetic beads: comparison with standard Percoll density gradient isolation methods

A prerequisite for studies on bovine neutrophils is a reliable method of neutrophil isolation from blood to obtain highly purified cell populations that are functionally active. Since current techniques of neutrophil isolation fall short of these requirements, we have developed a newer and more effective technique for isolation of bovine neutrophils that utilizes biomagnetic beads coated with a monoclonal antibody that recognizes an abundant surface antigen on bovine neutrophils to purify these cells. Comparison of the purity and viability of bovine neutrophils isolated by a conventional method (continuous Percoll density gradient) with this new method showed that neutrophils isolated with biomagnetic beads were higher in purity and had an increased yield. In addition, cells isolated with biomagnetic beads demonstrated normal or even improved function in assays of chemotaxis, phagocytosis, degranulation, and respiratory burst activity. Finally, bovine neutrophils isolated using this method showed an overall lower level of spontaneous apoptosis, which correlates well with the high level of viability observed in the purified cell preparations. Thus, this method represents a significant advance over current methods for isolating bovine neutrophils and would be widely applicable to labs studying the biochemistry and signal transduction pathways in these cells.

Rapid particle size distribution analysis of Bacillus spore suspensions

Knowledge of the homogeneity of a spore crop is an essential criteria for the subsequent investigation of their properties. Two methods of analysis have been compared; particle size distributions of Bacillus spores suspensions were analysed by laser light scattering with a Malvern Mastersizer; and scanning transmission electron microscopy (STEM) was used to record images of 50–100 individual spores. With an image particle sizing analysis software the dimensions (length, breadth, perimeter) of single spores were determined. Good agreement between the average individual spore dimensions determined by electron microscopy and those obtained by light scattering was evident. The Mastersizer was convenient and rapid to use but the image analysis provided the opportunity to distinguish between spores, vegetative cells and other particles. Spore suspensions were further separated from vegetative cells by centrifugation in continuous and discontinuous Percoll (modified colloidal silica) density gradients (1.09–1.13 g/ml). Electron microscopy revealed an adhesion of colloidal silica particles to the spore surface which could only be partially removed by repetitive washing. High silica contents were therefore detected by X-ray microanalysis. Percoll did not effect differential scanning calorimetric analysis of spores.

HeLa cells as a model to study the invasiveness and biology ofLegionella pneumophila

HeLa cells were established as a model system to study the invasiveness and biology of Legionella pneumophila. In this model, invasion could be distinguished from adherence; virulent strains of L. pneumophila were adherent and invasive, whereas nonvirulent strains were adherent but poorly invasive. Invasion was rapid and did not require de novo bacterial protein synthesis, suggesting that the invasion factor is constitutively expressed by virulent strains. Entry into HeLa cells required actin polymerization and an intact microtubule cytoskeleton and was only moderately inhibited by the presence of 100 mM glucose or galactose. Intracellular replication of virulent L. pneumophila took place in ribosome-studded complex endosomes and led to the formation of free bacteria-laden vesicles presumably released from lysed HeLa cells. These free vesicles (referred to as mature vesicles) were isolated in continuous density gradients of Percoll. The bacteria contained in the isolated mature vesicles had a unique envelope structure and were highly adherent to HeLa cells, characteristics that correlated with a bright red appearance after the Giménez stain (Giménez positive). Plate-grown legionellae and replicating legionellae, harboured in complex endosomes, displayed a typical Gram-negative envelope and stained green after the Giménez stain (Giménez negative). Chronically infected cultures of HeLa cells were also established that may be a useful tool for studying long-term interactions between virulent L. pneumophila and mammalian cells. HeLa cells constitute a valuable model system that offers unique opportunities to study parasite-directed endocytosis, as well as stage specific-parasite interactions.

No induction of chromosome aberrations in human spermatozoa exposed to extremely low frequency electromagnetic fields

Clastogenic effects of extremely low frequency electromagnetic fields (ELF-EMFs) on human sperm chromosomes were studied using an interspecific in vitro fertilization system with zona-free golden hamster oocytes. Semen samples from healthy men were exposed to ELF-EMFs (50 Hz, 20 mT) for 2 h at 37°C under 5% CO 2 in air. The samples were then cryopreserved in liquid nitrogen for shipment to a cytogenetic laboratory. After thawing the samples, motile spermatozoa were collected using a continuous Percoll density gradient centrifugation and then capacitated for in vitro fertilization with hamster oocytes. Sperm-derived chromosomes were analyzed at first cleavage metaphase. The present experiment was performed twice using semen samples from two different donors. In test-1, incidence of spermatozoa that displayed structural chromosome aberrations was 17.0% (35/206) in the exposed group and 20.8% (55/264) in the control group. In test-2, structural chromosome aberrations were observed in 11.1% (13/117) of exposed spermatozoa and 13.8% (13/94) of spermatozoa in the control group. In both tests, there was no significant difference in the incidence of chromosomally abnormal spermatozoa between the exposed group and the control group. Types of aberrations observed and their incidences per spermatozoon in the exposed group were similar to those of the control group. Despite the small sample size, the present results suggest that ELF-EMFs have no clastogenic effect on human sperm chromosomes.

The effects of cromakalim on the mediator releases from guinea pig lung mast cell activated by specific antigen-antibody reactions.

The inhibitory effect of cromakalim on the mediator release from mast cells caused by antigenantibody reactions was in controversy with the specific antigen used. However, it has recently been observed that cromakalim inhibits the release of mediators from superfused tracheal and parenchymal strips or lung mast cells after passive sensitization with the IgG1 antibody. An attempt, therefore, was made to determine the inhibitory mechanisms of cromakalim on the release of mediators such as histamine and leukotriene released by the activation of enzymes during mast cell activation. Guinea pig lung mast cells were purified through enzyme digestion, rough percoll and continuous percoll density gradients. The purified mast cells were prelabeled with [3H]palmitic acid. PLD activity was assessed more directly by the production of labeled phosphatidylethanol by PLD-mediated transphosphatidylation in the presence of ethanol. In the cells labelled with [3H]myristic acid, [3H] DAG production was measured. The methyltransferase activity was assessed by measuring the incorporation of [3H]methyl moiety into phospholipids in sensitized mast cells labelled with L-[3H] methylmethionine. cAMP level was measured by radioimmunoassay. Cromakalim resulted in a decrease in the amount of histamine and leukotrienes releases by 30% in the ovalumin-induced mast cell. Cromakalim had little effect on phospholipase D activity enhanced by the activated mast cell. Cromakalim inhibited the initial increase of diacylglycerol production during mast cell activations. Cromakalim inhibited the phospholipid methylation increased in the activated mast cell. These results show that cromakalim decreases histamine release by inhibiting the initial increase of 1,2-diacylglycerol during the mast cell activation, which is mediated via the phosphatidylinositide-phospholipase C system rather than the phosphatidylcholine-phospholipase D system. Furthermore, cromakalim reduces phosphatidylcholine production by inhibiting the methyltransferase, which decreases the conversion of phosphatidylcholine into arachidonic acid and inhibits the production of leukotrienes.

Phosphoprotein phosphatase activities in rat luteal cells

The phosphorylation and dephosphorylation of proteins is known to play an integral part in the metabolic processes of a cell 111. Whilst the importance of protein kinases and phosphorylation in regulating luteal cell steroidogenesis is widely accepted [2], the involvement of phosphoprotein phosphatases (PP) in the regulation of luteal cell function remains unclear. Of the four types of serinehhreonine PP's (PPl, PP2A, PP2B and PP2C) known to be active in cellular regulation 131, types PPI and PP2A are suggested to be involved in the regulation of intracellular mechanisms of secretory cells 141. The objective of this study was to determine the activity of PP's in rat luteal cells, and to examine the effects of inhibiting these enzymes. Female Sprague-Dawley rats (24-28 days old) were superovulated by sequential injections of pregnant mare serum gonadotrophin (Folligon, Intervet, U.K. Ltd. 50 IU) and human chorionic gonadotrophin (Chorulon, Intervet, U.K. Ltd. 25 IU) [ S ] . Whole luteinized rat ovaries were collagenasedigested and purified on continuous Percoll density gradients (40%) as previously described [S]. PPI and PP2A activity was assayed by measuring the release of '*P from 3ZP-radiolabelled glycogen phosphorylase a, using a commercially available in vitro assay kit (Gibco BRL, U.K.). The effects of PP inhibition on the dephosphorylation of endogenous substrates were examined by measuring 32P transfer from Y~~P-ATP, in vitro, onto endogenous luteal cell proteins, separated by SDS-polyacrylamide gel electrophoresis and visual ised by autorad iograph y . A range of structurally dissimilar inhibitors of PPI and PP2A were effective inhibitors (P PPI in vitro [3,7,81, inhibited PP activity with a minimum effective dose of 10'M, over the concentration range 10~'M to IO~'M. Cantharidin, which also has dissimilar sensitivity, effecting PP2A>PPI in vitro [9], showed a minimum effective dose of lO~M, over the concentration range

Hormonal storage patterns and morphological heterogeneity of porcine gonadotrope cells during postnatal development

Previous reports indicate that gonadotrope cells of the porcine pituitary gland can be separated into three subpopulations of low- (1.049 g/cm 3), middle- (1.062 g/cm 3) and high- (1.087 g/cm 3) density in a continuous Percoll density gradient. The aim of this work was to study the hormonal storage patterns and morphological features of these subpopulations at three representative ages of the postnatal development: neonatals (30-day-old animals), prepubertals (5–6-month-old animals) and matures (16–18-month-old animals). The low-density subpopulation, present at the three ages studied, was mainly composed of bihormonal LH/FSH cells in neonatal and monohormonal LH cells in prepubertal and mature animals. On the other hand, middle- (only present in prepubertal and mature animals) and high-density subpopulations (only present in neonatal and prepubertal animals) were mainly composed of bihormonal LH/ FSH gonadotropes. In ultrastructural terms, these subpopulations exhibit a correlation between density and morphology irrespective of the animal's age. The low-density subpopulation was composed of poorly granulated cells with highly developed biosynthetic machinery (rough endoplasmic reticulum and Golgi complex), while high-density cells were of opposite morphology, with a highly granulated cytoplasm and poorly developed rough endoplasmic reticulum and Golgi complex. The middle-density subpopulation was composed of poorly granulated cells with scarcely developed biosynthetic machinery. In conclusion, these results indicate that porcine gonadotrope cells during postnatal development are composed of three subpopulations of different hormonal storage patterns and morphology. The presence of these subpopulations at the different stages of postnatal development strongly suggests that their proportions may play a major role in the endocrine control process.

017 Enrichment of rat corticotrophic cells by centrifugation on a continuous Percoll density gradient: are there corticotrophic subpopulations?

017 Enrichment of rat corticotrophic cells by centrifugation on a continuous Percoll density gradient: are there corticotrophic subpopulations?

Heterogeneity in granulosa cells of developing rat follicles

The present study was designed to characterize the granulosa cell subpopulations derived from rats in which ovarian growth was induced by diethylstilbestrol (DES) or in which growth and differentiation was induced by pregnant mare's serum gonadotropin (PMSG). In the DES model, immature rats were given two separate injections of 2 mg DES/rat s.c. on 25 and 26 days of age and were killed 24 hr after the second injection. In the PMSG model, rats on day 28 were given 8 IU of PMSG s.c. and were killed 54 hr later. Granulosa cells were isolated from the ovaries, separated on a continuous Percoll density gradient, and divided into 12 fractions. The cells in each fraction were cultured in the presence of androstenedione with or without 20 ng/ml of follicle-stimulating hormone (FSH) and estradiol and progesterone in the incubation medium were measured. In DES-treated rats, granulosa cells in fractions 4 and 5 and fractions 9 and 10 contained about 30-40% of total cell yield and showed high steroidogenic potential. Granulosa cells from fractions 6, 7, and 8, which represent 55-60% of total cell yield, produced relatively low amounts of steroids on a per-million-cell basis. FSH was required for the stimulation of steroidogenesis. In granulosa cells from the PMSG treated rats, aromatase activity appeared maximally induced and incubation with FSH in vitro did not bring about any further increase. However, in vitro incubation with FSH was required for progesterone production. Furthermore, the granulosa cells from the PMSG treated rats also showed much more active estradiol and progesterone synthesis in fractions 9-12 as compared with lower density fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Subpopulations of primary adult murine epidermal basal cells sedimented on density gradients

Epidermal cells were harvested from the dorsal skin of adult mice by trypsinization and were sedimented through continuous density gradients of Percoll, formulated to separate basal cells of different buoyant density. Five fractions from the gradients were characterized with regard to the number of cells present, their viability and morphology and their basal origin. Suprabasal keratinocytes remained primarily at the top of the gradient; basal keratinocytes sedimented throughout. With increasing density, a relative enrichment was observed: (i) for [3H]-thymidine and [3H]-benzo[alpha]pyrene label-retaining (slowly cycling) keratinocytes; (ii) for keratinocytes that could proliferate in vitro in the continuous presence of 0.1 micrograms ml-1 of 12-O-tetradecanoylphorbol-13-acetate; (iii) for cells from untreated as well as initiated epidermis able to proliferate under conditions where calcium induces terminal differentiation; and (iv) for primary in vitro clonogenic keratinocytes from normal epidermis. The relative enrichment for epidermal basal cells having characteristics thought to be associated with immaturity and with the initiation and promotion of skin carcinogenesis suggests that density gradient sedimentation could be used in conjunction with other methods for the eventual purification of epidermal progenitors.

Isolation and morphologic differentiation in vitro of villous cytotrophoblast cells from rhesus monkey placenta

Trophoblast is the major functional cell type of the placenta. The purpose of this study was to devise a means to isolate trophoblast cells from the monkey placenta and to examine their capacity to differentiate in vitro. Methods originally devised for the isolation of human cytotrophoblast cells produced poor yields and a low degree of purity when applied to the near-term rhesus monkey placenta. However, a procedure has been developed which allows the isolation of a cell population consisting of more than 95% cytotrophoblast based on intermediate filament immunocytochemistry. The cells sedimented between densities of 1.040 and 1.053 g/ml on continuous Percoll density gradient centrifugation. When maintained in culture the cells adhered and formed aggregates of mononuclear cells by 24 h. By 5 d in culture, immunofluorescent staining using antidesmoplakin and antinuclear antibodies revealed that most colonies consisted of large multinucleated masses similar to syncytiotrophoblast. These results demonstrate trophoblast cells from monkey placental villi can be isolated with a high degree of purity and undergo morphologic differentiation in vitro. This preparation should enable investigators to study many functional characteristics of these cells throughout gestation.

Long-term culture of rat mammotrope and somatotrope subpopulations separated on continuous Percoll density gradients: Effects of dopamine, TRH, GHRH and somatostatin

Normal adult female rat mammotrope and somatotrope subpopulations were separated on continuous Percoll density gradients according to differences in their density. Viable cells were recovered in 16 fractions. The cells from each fraction were cultured during 7 days after which period 4-h incubations were performed. rPRL secretion per cell increased towards the higher density fractions. No major difference in TRH, dopamine and somatostatin responsiveness was observed between mammotropes that were recovered in the different gradient fractions. In addition, no differences in somatostatin responsiveness between the somatotrope cells in the different gradient fractions were observed. However, somatotropes that were recovered in the highest density region of the gradient appeared to be more responsive to GHRH than the lower density somatotropes. In the various gradient fractions there were no paradoxical effects of TRH and dopamine on rGH release and of GHRH on rPRL release. 1. In long-term cultures there is no evidence for functionally different subpopulations of mammotropes and somatotropes, separated according to differences in their density, with regard to dopamine and TRH responsiveness and with regard to somatostatin responsiveness, respectively. 2. There is no evidence for a (mammosomatotrope?) subpopulation of cells showing paradoxical responses of PRL or GH release to GHRH and dopamine or TRH, respectively.