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At least 1,531 records · Page 85Linked to original sources
PubMed1975

[Separation of ribonucleoprotein particles (Hn-RNP) from nuclei of L cells, in two classes, by isopycnic centrifugation in metrizamide gradients].

Separation of nuclear Hn-RNP particles into two density classes, following isopy cnic centrifugation in metrizamide gradients, is reported. One class (density 1.31 g/ml) is rapidly labelled, and contains polydisperse heterogeneous high molecular weight particles, easily diffusible from intact nuclei under certain conditions. The other class (density 1.18 g/ml) is of lower molecular weight, not diffusible, and needs apparently a longer time to be labelled and/or to accumulate inside the nuclei.

Animals↗
PubMed1984

High-pressure dissociation of the beta 2-dimer of tryptophan synthase from Escherichia coli monitored by sucrose gradient centrifugation.

The isolated beta 2-dimer of Escherichia coli tryptophan synthase exhibits reversible high-pressure deactivation and hybridization with an equilibrium transition at 690 and 870 bar for the apoenzyme and holoenzyme, respectively. To investigate the hypothetical dissociation mechanism ultracentrifugal analysis has been applied. In a conventional swing-out rotor (r(max) = 16 cm, fill-height 9 cm) a pressure gradient of 1 less than p less than 1840 bar is formed at maximum speed (40 000 rpm). Using a sucrose gradient to stabilize the particle distribution, pressure-dependent alterations of the state of association of oligomeric systems may be determined. In the present experiments ovalbumin (with a molecular mass close to the beta-monomer) has been used as a reference. The radial sedimentation velocity of the beta 2-dimer (in 5-20% sucrose, 10 degrees C) is found to decrease significantly at p approximately equal to 850 bar. From the slopes in an r-r(degrees) vs t plot the limiting values for the particle weight at the meniscus and the bottom of the tube are found to be the beta 2-dimer (M(r) = 85 800) and the beta-monomer (M(r) = 42 900), thus proving pressure-dependent dissociation. Since sucrose stabilizes the native quaternary structure, the beta 2 leads to 2 beta transition is shifted towards higher pressures compared to the dissociation in standard buffer. Conventional quench experiments in high-pressure cells in the presence of 13% (w/v) sucrose confirm the result of the sucrose gradient centrifugation with respect to the critical pressure where deactivation (and dissociation) occur.

Centrifugation, Density Gradient↗
PubMed1988

A centrifugation method for separation of plant viral genomic and subgenomic RNAs.

Using alfalfa mosaic virus (AMV) as a model, a simple method for separating plant viral genomic RNAs from their subgenomic counterparts was established. The method relies on sucrose gradient fractionation under carefully selected conditions of centrifugation and fraction collection. The RNA components are recovered in nearly quantitative yield and have full biological activity as measured by infectivity of the reconstituted RNAs in suitable protoplasts and plant hosts. The individual RNAs, on the other hand, show no such infectivity, indicating that the separation is indeed complete.

Centrifugation, Density Gradient↗
PubMed1982

A modified neutral sucrose centrifugation method for rapid detection of DNA damaged by gamma-radiation and repair in human lymphocytes.

1. The influence of 60Co gamma-radiation on the sedimentation behaviour of human lymphocyte nucleoids in neutral sucrose gradients was studied with the aid of a modified fluorescent monitor system consisting of the DNA-binding dye, Hoechst 33258 and a vertical centrifuge rotor. 2. A combined method employing the sedimentation procedure and the incorporation of tritiated nucleosides was utilized to evaluate the repair of gamma-radiated DNA.

Centrifugation, Density Gradient↗
PubMed1977

The effect of centrifugal force on glycosaminoglycan production by aortic smooth muscle cells in culture.

Cultured smooth muscle cells from pig aorta subjected to centrifugation (48 h at 45 g over a 72-h period) increased their production of glycosaminoglycans by approximately 50%. The sulphated components, heparan sulphate, dermatan sulphate and chondroitin sulphate, showed a relatively greater increase than hyaluronic acid (66-34%). The results are consistent with the hypothesis that mechanical stress, such as hypertension, leads to increased accumulation of glycosaminoglycans in the aortic wall and that secondary trapping of lipoproteins by sulphated glycosaminoglycans produces atherosclerotic plaques.

Animals↗
PubMed1992

Counter-current chromatography of lipoproteins with a polymer phase system using the cross-axis synchronous coil planet centrifuge.

Lipoproteins were separated by counter-current chromatography using the type-XLL coil planet centrifuge. The separation was performed with a polymer phase system composed of 16% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate by eluting the lower phase at a flow-rate of 0.5 ml/min. About 5 ml of the sample solution containing approximately 150 mg of a lipoprotein mixture were loaded. High- and low-density lipoproteins were resolved within 12 h. Each component was detected by gel electrophoresis with oil red staining.

Centrifugation↗
PubMed1993

Studies on a new cross-axis coil planet centrifuge for performing counter-current chromatography. I. Design of the apparatus, retention of the stationary phase, and efficiency in the separation of proteins with polymer phase systems.

An improved model of the cross-axis synchronous flow-through coil planet centrifuge has been designed in light of previous studies. The apparatus has a versatile feature in that both analytical and preparative columns can be accommodated in both off-center and central positions. Each has merit in separations. Retention of stationary phase was examined with various two-phase solvent systems used for the separation of biopolymers. Both analytical and preparative columns showed satisfactory retention of the stationary phase under optimum conditions. The apparatus was evaluated in separation of a set of protein samples using a polyethylene glycol-potassium phosphate biphasic system. In both types of columns all proteins were resolved with partition efficiencies of 260 to 670 theoretical plates. Further studies indicated that the relatively low partition efficiency of proteins is mainly attributed to their high molecular mass or molecular heterogeneity within each species rather than due to the high viscosity of the polymer phase system.

Centrifugation↗
PubMed1993

Studies on new cross-axis coil planet centrifuge for performing counter-current chromatography. III. Speculations on the hydrodynamic mechanism in stationary phase retention.

Retention of the stationary phases of one organic-aqueous solvent system and three aqueous-aqueous polymer solvent systems was investigated on a cross-axis coil planet centrifuge. A graphic statistical treatment of all the data highlighted the prevailing effect of the inward-outward elution mode. A simplified model was proposed and studies on the paths and accelerations of cross-axis devices described in the previous paper provided explanations about the observed hydrodynamic behaviors.

Centrifugation↗
PubMed1994

Revealing surface changes associated with maturation of ram spermatozoa by centrifugal counter-current distribution in an aqueous two-phase system.

Centrifugal counter-current distribution (CCCD) in an aqueous two-phase system was used to detect changes associated with maturation of ejaculated ram spermatozoa. Spermatozoa obtained from three successive ejaculates of rams maintained in abstinence for one, two and three days were fractionated by CCCD. The results show that these ejaculates are relatively enriched in a cell population which presents a very high enhanced affinity to the lower dextran-rich phase. This cell population is not associated with loss of acrosomal integrity. In addition, it tends to disappear with longer abstinence periods, or after successive ejaculations at the same abstinence period, strongly suggesting that it is composed of immature cells. Therefore, phase partitioning can detect surface changes accompanying sperm maturation and offers a new possibility for sperm quality analysis.

Animals↗
PubMed1977

Immuno-ultracentrifugation: a combination of density gradient centrifugation and immunodiffusion.

By incorporating acrylamide in a sucrose gradient and photopolymerizing the tubes at the end of the run, direct immunochemical and other analysis can be performed on the separated material in situ without recovering fractions from the centrifuge tube. Immunodiffusion performed on the sliced gel can be employed to locate antibody activity in the gel slice or determine the distribution of antigens in a sucrose gradient

Animals↗
PubMed1979

Non-selective lymphocyte isolation from human blood by nylon wool filtration and density centrifugation.

A new method was employed to isolate lymphocytes from human peripheral blood. Continuous flow filtration through a nylon wool filter, at a flow rate of 1.4 ml/min, produced a lymphocyte yield of 90.5% and a purity of 96% without any shift in the B-T cell ratio. Ficoll-Isopaque with a specific gravity of 1.085 g/ml instead of 1.077 g/ml could be used to remove the erythrocytes. An overall recovery, including defibrination, filtration, Ficoll-Isopaque centrifugation and washing step, of 74.5% was achieved.

B-Lymphocytes↗
PubMed1980

Isolation of human T and B lymphocytes by E-rosette gradient centrifugation. Characterization of the isolated subpopulations.

Some methodological aspects of E-rosette gradient centrifugation for separation of human B and T lymphocytes are described. Treatment of sheep red blood cells (SRBC) with 2-aminoethylisothiouronium bromide (AET) accelerates and enhances E-rosette formation providing an effective and time saving method which takes less than 2 h to perform. Depletion of E-rosette forming cells (E-RFC) is almost complete, leaving less than 1% of the initial E-RFC at the interphase layer. By use of the lymphocyte donor's autologous serum containing naturally occurring anti-SRBC antibody and complement the SRBC in the rosetted T lymphocyte fraction are easily and completely lysed without damage to the lymphocytes. This procedure is shown to have little effect on the properties of the isolated T cells. The isolated lymphocyte subpopulations have been characterized by tests for lymphocyte and monocyte markers.

B-Lymphocytes↗
PubMed1980

Isolation of human NK cells by density gradient centrifugation.

Sedimentation characteristics of human NK cells in discontinuous Percoll density gradients were studied. NK activity against the leukaemic cell line K-562 peaked in a single low density fraction mainly consisting of large granular lymphocytes previously shown to be the principal NK cells in human peripheral blood. Percoll density gradient centrifugation provides a useful tool for analysis of human NK cells and their relationship to other blood mononuclear cells.

Cell Fractionation↗
PubMed1980

A rapid single centrifugation step method for the separation of erythrocytes, granulocytes and mononuclear cells on continuous density gradients of Percoll.

A simple rapid method of separating mononuclear leucocytes, granulocytes and erythrocytes on continuous Percoll density gradients has been developed. The Percoll is mixed with increasing amounts of the blood, which forms the light phase of the gradient. Centrifugation at 400 X g for 30 min results in almost complete separation of the 3 groups of cells with high recoveries and degrees of purity. Blood samples from 3 patients with chronic granulomatous disease were separated with this technique and the pattern of cell separation was found to be different from normal.

Adult↗
PubMed1981

Separation of AKR mouse thymus lymphoma from normal thymic cells by centrifugal elutriation.

The rapid separation of large numbers of viable thymus cells from AKR mice bearing transplanted or spontaneous thymic lymphomas was achieved by centrifugal elutriation. Separation of more than 3 x 10(8) cells from either a transplanted lymphoma, designated 720, or from a spontaneous thymic lymphoma, required only 15 min. Unfractionated thymus cells obtained from mice bearing the transplanted lymphoma consisted of 80% lymphoma cells (by immunofluorescence for the viral protein, gp70) and 20% normal cells. Fractions of slowly sedimenting cells consisted almost exclusively of normal cells (95%) with modal volumes of 95 micrometers cubed. Fractions of rapidly sedimenting cells consisted of 95% tumor cells with volumes of 150-400 micrometers cubed. The slowly sedimenting cells were almost exclusively (98%) in the GO- or G1-phase. Fractions of rapidly sedimenting cells contained up to 55% S-phase and up to 30% G2-phase cells. Intermediate fractions contained mixtures of normal cells and small GO- or G1-phase tumor cells. Thymidine uptake by the separated cells was determined. The fractions containing normal cells showed little thymidine uptake after 4 and 48 h in culture, while the fractions of tumor cells showed high levels of incorporation. In contrast to the high levels of thymidine uptake by the tumor cell fractions after 48 h in culture, there was little uptake by the unseparated cell suspension, suggesting a possible interaction between normal and tumor cells during the culture period.

Animals↗
PubMed1982

A single-step centrifugation method for separation of granulocytes and mononuclear cells from blood using discontinuous density gradient of Percoll.

A rapid and reproducible method is described for the simultaneous purification of mononuclear and polymorphonuclear cells, based on the use of a discontinuous gradient of Percoll. Centrifugation at 600 X gav for 20 min resulted in separation of the mononuclear and polymorphonuclear cells into 2 distinct bands at the interfaces. The upper layer contained the mononuclear cells and the lower band highly purified polymorphonuclear cells. Both populations were purified about 97% without detectable cell alteration.

Cell Separation↗
PubMed1983

Separation of mouse lymphoblasts by discontinuous density centrifugation on Percoll gradients.

Mouse lymphoblasts generated in vivo by a topical application of the contact sensitizer oxazolone or by the contents of the gut lumen were separated by discontinuous density centrifugation on Percoll gradients. A 3-step gradient was used to divide the cells into two subpopulations. For cells from oxazolone stimulated lymph nodes, the low density band contained 20-30% of the initial cell number applied to the gradient; 25-40% of this population were in S phase and nearly all the large and pyroninophilic cells were confined to this layer. The high density step cells (70-80% of initial cell number) were predominantly small lymphocytes with less than 0.5% in S phase. Similar results were obtained using cells from picryl chloride stimulated lymph nodes or from mesenteric lymph nodes.

Animals↗
PubMed1983

A single centrifugation step method for the simultaneous separation of different leukocytes with special reference to basophilic leukocytes.

A simple method of separating mononuclear leukocytes, basophils, neutrophils and an eosinophil fraction from human blood is described, using discontinuous Percoll gradient centrifugation. Mononuclear cells were found in the upper layer (band 1) at a density of less than or equal to 1.072. The second band with a density of less than or equal to 1.082 contained most of the basophilic leukocytes (av. 70% and 8-20% of the differential cell count) and a minor part of the mononuclear cells (av. 8%). In the third band, density less than or equal to 1.1, 82-91% of the polymorphonuclear cells (PMN) were found, consisting mainly of neutrophils. 35-70% of the eosinophilic PMN cells are located in band four (density greater than 1.1). The function of the basophil-enriched fraction was tested by degranulation with specific antigen.

Basophils↗