Density Gradient Medium Research Articles

Sedimentation-Flotation Focusing Field-Flow Fractionation in Channels with Modulated Cross-Sectional Permeability. II. Experimental Implementation

Abstract The first experimental separation of the model particles according to their differences in densities by a new Sedimentation-Flotation Focusing Field-Flow Fractionation (SFFFFF) method is described. The SFFFFF was accomplished in a simple fractionation channel by using a density gradient medium and applying the natural gravitational field of 1 G. Although the experimental results are of the preliminary character, they proved decisively the real potential of the SFFFFF.

The combined procedures of Ficoll-Paque centrifugation and anion exchange separation for the recovery of trypanosomes from blood and other tissues

The conventional method of separating African trypanosomes from infected blood using a DEAE-cellulose (Whatman Chromedia, DE-52) column was modified by the inclusion of an initial centrifugation step employing the density gradient medium, Ficoll-Paque. On centrifugation in this medium using diluted infected rodent blood the trypanosomes moved into the buffy coat interphase which consisted mainly of lymphocytes with a few contaminating RBCs and neutrophils. The buffy coat interphase layer was withdrawn, washed, resuspended and the trypanosomes finally separated from the contaminating blood cells by passage through buffer-equilibrated DE-52 packed in a 25 ml syringe barrel, representing a considerable saving of DE-52 over the conventional method. The centrifugation step was applied to the investigation of other tissues of infected rodents, such as bone marrow, brain, spleen, liver, lymph nodes, kidney and testis, for the presence of trypanosomes. Using the combined procedure, high recoveries of purified brucei group trypanosomes were obtained from bone marrow, brain, spleen and lymph nodes.

Purification of peptides derived from proenkephalin A on Bio-Rex 70

The weakly acidic, cation-exchange resin Bio-Rex 70 was used at a low pH for chromatography of enkephalins and other peptides containing enkephalin sequences. Recoveries of various peptide standards and peptides in adrenal chromaffin cell extracts were greater than 85%. Catecholamines and other substances such as density gradient media are not retained by the resin under the conditions employed. A significant purification of enkephalins and larger peptides containing enkephalin sequences with respect to total protein was also obtained by this method. This chromatographic technique should be useful where a low-cost, preliminary purification of samples containing peptides derived from proenkephalin A is required and may be adaptable to other peptides as well.

Purification and infectivity ofEntomophaga grylli (Fresenius) Batko Pathotype 2 againstMelanoplus differentialis (Thomas) [Ort.: Locustidae

The life stages ofEntomophaga grylli (Fresenius) Batko Pathotype 2 were purified and separated by centrifugation in PercollR density-gradient medium. The ranges of buoyant densities for germinated resting spores, germ conidia, and resting spores respectively were: 1.040–1.050, 1.055–1.085, and 1.080–1.120 g/ml. Cuticular invasion by germinated germ conidia was the means by whichMelanoplus grasshoppers became infected. Scanning electron micrographs revealed germination of germ conidia on the visible host integument at 100% RH, but not at 90% RH. Significantly higher mortality (P<0.05) was obtained after 3 weeks with grasshoppers incubated in constant light than in constant dark for 24 h following treatment. The disease was not transmitted by ingestion of any life stage.

Caractérisation et différenciation de préadipocytes de rats en culture primaire

Using a density gradient medium, we isolated homogeneous cell populations from the inguinal tissue of 3-day old rats. In primary culture we obtained, for the first time, the differentiation of 99% of the cells in the presence of a physiological concentration of insulin (10(-9) M). This model closely mimicked the events occurring during normal mammalian adipose development, i.e. a positive change in beta-adrenergic sensitivity, early induction of lipoprotein lipase, expression of the enzymes involved in the triglyceride systems, and the development of responsiveness to glucagon.

Effects of Polymethylmethacrylate on Rabbit Articular Chondrocytes in Monolayer Culture

The effects of polymethylmethacrylate (PMMA) on DNA, protein, and sulfated-proteoglycan synthesis by rabbit articular chondrocytes were observed in monolayer cultures. PMMA pellets in ratios of 1:1 and 1:2 (liquid monomer:powder) significantly reduced [3H]thymidine incorporation into DNA during the first 24 h of culture and less so after 48 and 72 h. The reduction in [3H]thymidine incorporation was restricted to the cohort of chondrocytes nearest the PMMA. Consequently, cellular proliferation was unaltered by PMMA. By contrast, PMMA failed to inhibit [3H]leucine or [3H]serine/35SO4 incorporation. Both control and PMMA (1:1)-treated chondrocyte CsCl density gradient medium fraction dA1 eluted as a retarded peak on Sepharose CL-2B under associative conditions. The average partition coefficient (Kav) of PMMA-treated fraction dA1 was 0.41, as compared with 0.27 for control cultures. The Kav of medium fraction dD1 (proteoglycan monomer) was unaltered. Both control and PMMA-treated dA1 fraction elution profiles on Sepharose CL-2B were altered by incubation with Streptomyces hyaluronidase, indicating the presence of proteoglycan aggregate. The PMMA-treated cultures synthesized smaller proteoglycan aggregates. Since PMMA has been a critical factor in the success of total joint arthroplasty, defining interactions of differentiated cells with the cement is imperative for an understanding of the effects of PMMA on the biology of cartilage and bone.

Isolation of bovine polymorphonuclear leukocytes by density gradient centrifugation

A method for the isolation of an enriched population (> 95%) of bovine polymorphonuclear leukocytes (PMNs) was developed using density gradient centrifugation. Leukocytes were isolated from peripheral blood by centrifugation in a density gradient medium (Percoll) of specific gravity 1.092. Viability was ≥95% and the isolated PMNs were functional in migration inhibition and chemiluminescense assays. This has proved to be a simple effective method for obtaining bovine PMNs and yields cell populations that can be utilized for a variety of measures of PMN function.

Differentiation under the control of insulin of rat preadipocytes in primary culture: Isolation of homogeneous cellular fractions by gradient centrifugation

Using a density gradient medium (Percoll) we succeeded in isolating homogeneous cell populations from the stromal-vascular fraction of the inguinal tissue of 3-day-old rats. In primary culture, in medium 199 supplemented with 10% fetal calf serum and 5.5 mM glucose, almost complete differentiation (90%) of these fractions was obtained for the first time in presence of a physiological concentration of insulin (10 −9 M). During the adipose conversion, insulin markedly enhanced the activities of glycerol-3-phosphate dehydrogenase and acid:CoA ligase. When VLDL and heparin were added with insulin to the medium, this effect was not potentiated. On the contrary, VLDL and heparin in presence of insulin increased the triglyceride content of the cells. With VLDL and heparin only, the biochemical and morphological characteristics of the cells were very similar to those observed in control culture. The heavier fraction was morphologically heterogeneous and did not undergo the adipose conversion to the same extent as the two lighter fractions. It was concluded that this model could be helpful in studying the proliferation and the differentiation of preadipocytes at an early stage of development.

Buoyant densities of macromolecules, macromolecular complexes, and cell organelles in Nycodenz gradients

Nycodenz is a new nonionic iodinated density gradient medium which has several advantages over metrizamide. Although, overall, biological samples band at similar densities in Nycodenz and metrizamide gradients, a number of significant differences were found. As compared with metrizamide, not only does Nycodenz appear to interact less with proteins but also the buoyant density of chromatin is less affected by the amount loaded onto the gradient. A high degree of resolution is obtainable using Nycodenz gradients; thus, it is possible to separate densitylabeled DNA and to subfractionate subcellular membrane fractions.

Separation of proteins, nucleic acids and nucleoproteins on Nycodenz, a new non-ionic, iodinated, density-gradient medium

Separation of proteins, nucleic acids and nucleoproteins on Nycodenz, a new non-ionic, iodinated, density-gradient medium

Nycodenz: A new nonionic iodinated gradient medium

The properties of Nycodenz, a new nonionic iodinated density gradient medium, are described. This compound shares a number of characteristics with its related compound metrizamide. However, the stabler, more inert nature of Nycodenz endows it with a number of favorable properties as a gradient medium.

Superoxide Dismutase (SOD) in Mouse Red Blood Cells Infected with Plasmodium berghei

Four zones were consistently seen after centrifugation. Each zone was removed separately with a pasteur pipet and washed 3 times with KGS by centrifugation (800 g for 10 min). Blood films from each zone were made on coverslips and stained with Leishman's stain. Reported here is a summary of three experiments (Table I). Zone 1 (top) consisted of cellular debris, hemozoin, and degenerate free parasites. Most noteworthy were zones 2 and 3. The greatest concentration of late developmental stages virtually free of uninfected erythrocytes were observed in zone 2. Zone 3 had a somewhat lower parasitemia but a very high percentage of trophozoites and schizonts. The majority of the ring forms were found in zone 4 along with a large number of trophozoites. The trophozoites and schizonts isolated from zones 2 and 3 have been used for immunological and biochemical studies. Parasites from all zones, except zone 1, were put back into culture where the growth and multiplication of the parasites was observed to be normal. Zone 4 was routinely placed back into culture and processed after 24 to 30 hr of incubation to yield an additional harvest of trophozoites and schizonts. Percoll has many advantages over other density gradient media. Its easy handling and exceptional properties make it useful for the concentration and isolation of large numbers of P. falciparum trophozoites and schizonts. The versatility of Percoll should allow for further modifications of this procedure to yield a single zone of trophozoites or schizonts.

Separation of Non‐filamentous Micro‐organisms from Soil by Density Gradient Centrifugation in Percoll

Soil suspensions were homogenized, and desorbed non‐filamentous micro‐organisms were concentrated in a minimum volume of buffer by low speed centrifugation. The cells were separated from inanimate material by flotation at the interface between the buffer and a silica sol/polyvinyl pyrrolidone density gradient medium (Percoll). Cell suspensions were removed from the interface and fractionated according to density by high speed centrifugation on discriminating density gradients in Percoll.Preliminary experiments indicated that most non‐filamentous soil micro‐organisms had densities in the range 1.081–1.123 g%sol;ml while Rhizobium isolated from crushed root nodules on Percoll was split into two bands of densities 1.081–1.110 and 1.041–1.073 g/ml. The lighter cells were the more pleomorphic.The efficiency of extraction of cells from soil was governed by the extent of their desorption from inanimate particles. As rigorous desorption procedures damage cells, extraction efficiencies were low; 10–20% of cells counted microscopically in soil were recovered from density gradients. Electron microscopy of soil micro‐organisms isolated by this method showed an unusual range of surface ornamentations on cell‐like structures of bacterial dimensions.

Separation of plant cell organelles by zonal centrifugation in reorienting density gradients

Preparative separations of plant cell organelles (glyoxysomes, proplastids, mitochondria, and endoplasmic reticulum) in an ordinary refrigerated centrifuge were obtained by sucrose density gradient centrifugation in a zonal rotor loaded and unloaded in the static manner. The quality of the separation which was monitored by marker enzymes and electron microscopy compares to analytical separations in swinging-bucket rotors. Membrane alterations observed in glyoxysomes and mitochondria are traced back to sucrose as a major component of the homogenization and density gradient medium.

Plasmodium yoelii and Plasmodium berghei: Isolation of infected erythrocytes from blood by colloidal silica gradient centrifugation

Percoll (colloidal silica coated with polyvinylpyrrolidone) and Ficoll (MW 400,000) were used to separate erythrocytes infected with Plasmodium yoelii and Plasmodium berghei from uninfected red blood cells. Samples of blood collected from mice in different phases of malarial infection were overlaid on cushions of 55% Percoll, 20% Ficoll, or 28% Ficoll, respectively, centrifuged, and the interphase layers compared. The best yield of parasitized erythrocytes (PE) was achieved using Percoll when about 95% of the erythrocytes infected by the late developmental forms of the parasites (late trophozoites, schizonts, and gametocytes) were recovered from the gradient interphase, irrespective of the phase of the infection and the number of young erythrocytes in the sample. No alteration of antigenicity (assessed by immunofluorescence) or of osmotic fragility (over the range of 160–460 mOsm) could be detected in PE separated by Percoll or by Ficoll. In addition, parasites separated on Percoll gradients showed no significant ultrastructural changes and retained their normal infectivity to mice. Although both gradient media could be used for the separation of Plasmodium-infected erythrocytes, Percoll presented some advantages over Ficoll. Apart from the better reproducibility of the separation of high yields of very pure PE obtained with Percoll, its lower viscosity allowed easier handling, and lower centrifugal forces were needed to enable the cells to reach their isopycnic positions. Thus, Percoll fulfilled many of the criteria for an ideal density gradient medium. Parasitized erythrocytes were isolated by an easy, reproducible, and inexpensive procedure, and separated cells retained their normal structure, antigenicity, and infectivity.

Matrix vesicles in chicken epiphyseal cartilage. Separation from lysosomes and the distribution of inorganic pyrophosphatase activity.

The extracellular matrix vesicles from epiphyseal cartilage of chickens were isolated by differential centrifugation. The matrix vesicles obtained showed considerable activity of lysosomal enzymes. This appears to have been due to lysosomal contamination because when we used a new density gradient medium (Percoll), the lysosomal enzyme activities and the activity of alkaline phosphatase could be totally separated. Electron microscopy of the alkaline phosphatase-rich fraction showed matrix vesicle-like structures. Phosphatase activities of the cells and matrix vesicles were further studied by Sephadex G-200 gel filtration. Specific magnesium-activated inorganic pyrophosphatase, distinct from nonspecific alkaline phosphatase, could be demonstrated in the cellular fraction. No such separate activity could be demonstrated in the matrix vesicle fraction, and it is supposed that the pyrophosphatase activity in the matrix vesicles originates from the alkaline phosphate.

The viability of cells grown or centrifuged in a new density gradient medium, Percoll(TM)

A new density gradient medium, Percoll (a modified colloidal silica), has been tested for toxicity in primary cultures of rat liver and calf testicle cells, and in continuous cultures of pig kidney and HeLa cells. The presence of Percoll did not appreciably affect the growth or viability of the cells as judged from cell counts and morphology. The various cells were also centrifugea in gradients of Percoll and subsequently cultured. The in vitro growth of the cells was similar to that of untreated cells. Rat liver cells were labelled in vivo with [ 125I]asialoceruloplasmin (parenchymal cells) or heat-denatured [ 125I]albumin (non-parenchymal cells). After dispersion of the cells and iso-pycnic centrifugation in Percoll the non-parenchymal cells banded preferentially at a lower density (1.04−1.05 g/ml) than parenchymal cells (1.07−1.09 g/ml). The two types of cells showed very different morphology in cell culture. The non-parenchymal cells retained their phagocytic properties during culture. Injured cells and cell debris band at the top of the Percoll gradients in contrast to their behaviour in gradients containing low molecular weight substances. Centrifugation in Percoll can be used to enrich viable cells.

Biological Separations in Iodinated Density Gradient Media

Biological Separations in Iodinated Density Gradient Media

SV 40 Nucleoprotein complexes: Structural modifications after isopycnic centrifugation in metrizamide gradients

Nucleoprotein complexes (NPCs) containing viral DNA and cellular histones except H 1 can be isolated from permissive cells infected with SV 40 [ 11 or polyoma virus [2]. Similar complexes can be obtained by mild dissociation of SV 40 or polyoma virions [3,4]. Electron microscopic studies of these complexes showed the presence of 2 1 * 2 v bodies or nucleosomes interconnected by naked DNA filaments [5,6]. This beaded structure is very similar to that of cellular chromatin [7,8]. The purification of viral NPCs, essentially limited to sucrose density gradients, is insufficient. Buoyant density centrifugations of irreversibly fixed NPCs have been described [91 l] , but the fixation process might introduce artifacts, and the material so purified is of little use for further biochemical studies. Metrizamide, a new density gradient medium [ 121, was recently used for the purification of chromatin in the absence of aldehyde fixation [ 131 , for the isolation of reconstituted DNA-histone complexes [ 141, for the purification of ribonucleoprotein particles [ 151, and for biochemical studies on polyoma NPCs [16]. We have isolated SV 40 nucleoprotein complexes by sedimentation on sucrose gradients, and further purified them by isopycnic centrifugation in metrizamide gradients. Detailed electron microscopic studies of SV 40 NPCs show that although the beaded circular structure is generally well preserved

Studies on the Proteinaceous Subcellular Particles in Rice Endosperm: Electron-Microscopy and Isolation

Electron-microscopic examination of rice endosperm revealed the existence of protein-aceous subcellular particles, 1 to 4 µ in diameter and spherical or oval in shape. Isolation of the particles was effected by differential centrifugation in density gradient medium after mechanical or enzymic disintegration of endosperm cells. The isolated particles were predominantly composed of protein, and residual constituents were mainly lipid and carbohydrate. Their shape and behaviors were similar to those found in the endosperm. These facts show that the subcellular particles concerned are “protein bodies” There seemed to be several kinds of protein bodies different with respect to their protein and lipid contents.