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At least 865 records · Page 48Linked to original sources
PubMed1995

Conductometric study of erythrocytes during centrifugation. I. Size distribution of erythrocytes.

Sedimentation of hardened erythrocytes in a centrifugal field was studied by time recording of the current chamber in the longitudinal and the transversal directions relative to the cells' movement. The results clearly indicate the existence of an erythrocyte concentration profile during centrifugation. The rates of both longitudinal and transversal current alteration increase with centripetal acceleration and with falling cell concentration. The pellet formed from hardened cells represents virtually incompressible body. It is shown that erythrocyte shape affects the pellet conductivity. Analysis of the data using the modified Stokes' law enables calculation of the cell size distribution. The modal size of macrocytes, normal erythrocytes and two samples of microcytes thus measured was 3.40, 3.01, 2.63 and 2.83 microns, respectively. These data demonstrate that conductometric analysis is useful for investigating abnormalities in erythrocyte size.

Blood Sedimentation↗
PubMed1980

Proteoglycan complex and proteoglycan subunit polydispersity. Study by isopycnic centrifugation in cesium sulfate density gradients.

A true isopycnic centrifugation method for the study of the bovine nasal cartilage proteoglycan polydispersity is presented. The use of cesium sulfate as gradient forming salt instead of cesium chloride allowed proteoglycan banding without any sedimentation at the bottom of the centrifuge tube. Apparent buoyant densities of proteoglycan monomer and proteoglycan aggregate were different. The present method provides a useful tool for the study of proteoglycan polydispersity and also allows us to follow the distribution of the link proteins in different proteoglycan extracts.

Amino Acids↗
PubMed1983

Rapid purification of plasmid DNA by a single centrifugation in a two-step cesium chloride-ethidium bromide gradient.

A procedure for rapid, preparative purification of plasmid DNA is described and compared with a conventional equilibrium centrifugation method. A discontinuous, two-step CsCl-ethidium bromide gradient is used, with the starting position of the plasmid-containing extract being at the bottom of the tube. During centrifugation in a fixed angle rotor, covalently closed circular plasmid DNA is separated from contaminating protein, RNA, and chromosomal DNA in 5 hr. Plasmids purified by this method are considerably less contaminated with RNA than when purified by a 48-hr equilibrium run in a homogeneous gradient, as determined by agarose gel electrophoresis and 5'-end-labeling studies. Plasmid DNA purified in two-step gradients can be used directly for restriction endonuclease analysis and DNA sequencing.

Autoradiography↗
PubMed1986

Differences in the cellular location of substances endocytosed by rat liver as observed from the distribution patterns obtained after isopycnic centrifugation in a sucrose gradient.

We have investigated the distribution of several substances endocytosed by rat-liver, after isopycnic centrifugation in a sucrose gradient of the MLP fractions (de Duve, Pressman, Gianetto, Wattiaux and Appelmans (1955) Biochem.J. 63, 604-617) isolated at increasing times after injection. It has been observed that there are changes in the distribution pattern with time depending on whether the substance is taken up by parenchymal or sinusoidal cells. The results suggest that centrifugation experiments can be informative with respect to the cellular location of a molecule endocytosed by the liver.

Animals↗
PubMed1986

Subcellular fractionation of liver organelles from phenobarbital-treated rats by counter-current partition and sucrose gradient centrifugation.

Counter-current partition, sucrose density gradient centrifugation and enzymic analysis were used to explore the changes in rat liver organelles induced by phenobarbital. There was a small increase in partition coefficient and marker enzyme activity of the endoplasmic reticulum. The modal density of the marker enzyme showed a significant decrease reflecting the proliferation of the smooth endoplasmic reticulum. The mitochondria showed a bimodal distribution with a small reduction of marker enzyme activity. In contrast, lysosomes and peroxisomes were relatively unaffected by phenobarbital treatment. Phenobarbital caused a small but statistically insignificant increase in gamma-glutamyl transferase activity: density gradient centrifugation studies indicated that the increased activity was predominantly in the biliary canalicular elements. In contrast, cytosolic gamma-glutamyl hydrolase activity was strikingly reduced by phenobarbital treatment.

5'-Nucleotidase↗
PubMed1984

Isolation of human mononuclear cell subsets by counterflow centrifugal elutriation (CCE). I. Characterization of B-lymphocyte-, T-lymphocyte-, and monocyte-enriched fractions by flow cytometric analysis.

Rapid separation of large numbers of human peripheral blood mononuclear cells into fractions enriched for B lymphocytes, T lymphocytes, or monocytes was accomplished by counterflow centrifugal elutriation (CCE). The first fraction contained 98% of the platelets. Ten additional fractions containing subpopulations of mononuclear cells were collected by sequential increases in the flow rate while maintaining a constant centrifuge speed. Analysis of the fractions using monoclonal antibodies revealed that fraction 2, which was free of esterase-positive monocytes, was highly enriched for B cells. T lymphocytes (OKT3+) were the predominant cell type found in fraction 4. No enrichment for T-lymphocyte-helper (OKT4+) or -suppressor (OKT8+) subpopulations was observed in the lymphocyte containing fractions. Three fractions (7-9), highly enriched for esterase-positive cells, were predominantly OKM1+ monocytes with no evidence of selective separation of monocyte subpopulations. Thus, cell fractions enriched for B cells, T cells, and monocytes could be obtained, by utilizing CCE, in large enough quantities to enable analysis of their functional properties. Of particular interest was the ability to separate small, resting B lymphocytes from monocytes.

Antibodies, Monoclonal↗
PubMed1987

Improved method for determination of high density lipoprotein cholesterol using a sensitive reagent and a centrifugal analyzer.

Enzymic measurement of high density lipoprotein cholesterol (HDL-c) using a sensitive reagent and a centrifugal analyser is described. The Boehringer Mannheim cholesterol esterase/oxidase reagent has been modified by the inclusion of 2,4,6-tri-bromo-3-hydroxybenzoic acid (TBHBA) which reacts with hydrogen peroxide and the 4-aminophenazone/peroxidase system to produce a quinone-imine dye with a four-fold greater molar absorptivity than that produced with phenol. The resulting reagent system has been developed for use with a centrifugal analyzer for the determination of plasma HDL fractions isolated with polyethylene glycol 6000, for which a reagent of high sensitivity is required. The method is linear to 4 mmol/l of HDL-c and between-run and within-run CVs ranged from 1.01-2.54%. Reagent costs are currently $US 0.12 per test and large numbers of assay samples can be processed rapidly and conveniently. The mean (+/- SD) HDL-c value for men was 1.09 (+/- 0.33) and for women, 1.35 (+/- 0.37) mmol/l.

Centrifugation↗
PubMed1983

Comparison of differentiating Dictyostelium discoideum cell types separated by an improved method of density gradient centrifugation.

During the migrating slug stage of development of the cellular slime mold Dictyostelium discoideum, the precursor cells of the mature spores and stalk are known to differ from each other. In order to explore these differences on a variety of levels, we have improved existing methods for the isopyknic purification of the precursor types. Prespore and prestalk amoebae obtained from mechanically dissociated slugs were separated by centrifugation on preformed Percoll density gradients. The isolated precursor cells are greater than 90-95% pure, show good viability and developmental competence, and can be obtained in relatively large amounts. We have performed initial comparisons of the differentiating amoebae with respect to their location within the slug, protein composition, and developmental capabilities. Density centrifugation reveals heterogeneity among posterior slug cells: The majority of these cells have the expected prespore characteristics, but there is also a minor (lighter) density fraction resembling anterior, prestalk amoebae. Thirty polypeptides synthesized preferentially by prespore or prestalk slug cells were detected by two-dimensional gel electrophoresis and fluorography. Some of these proteins are equally cell-specific when their accumulation is measured by silver staining; others are remarkably non-specific when stained. When the isolated precursor populations were allowed to complete development, they differed in terms of developmental territory size and timing, and in ways which suggest differences in cell cohesion and chemotaxis.

Cell Separation↗
PubMed1986

Disintegration of nucleoskeletal elements by metrizamide/2 M salt isopyknic centrifugation.

Supramolecular structures that remain bound to chromosomal DNA under high salt conditions are believed to anchor DNA in the interphase nuclear skeleton. In order to identify these anchorage structures, the non-DNA materials that remain firmly bound to chromosomal DNA under conditions that disintegrate the high salt-stable architecture of nuclei were investigated. Nuclei of Ehrlich ascites cells were histone-depleted by treatment with 2 M salt. The residual halo structures were gently sheared and subjected to metrizamide isopyknic centrifugation in the presence of 2 M salt. By this combined treatment the high salt stable nuclear skeleton becomes disintegrated and three main fractions are resolved. A light fraction comprises the DNA which appears to be essentially depleted of other nuclear components. The only non-DNA material that could be identified in the DNA band is a fraction of (nascent) RNP. No other materials which could reflect nucleoskeletal elements (e.g. lamina proteins) were found together with DNA. A peak of intermediate density comprises RNA/RNP dissociated from DNA. The heavy fraction contains the proteins that become dissociated from DNA by high-salt and/or centrifugal forces, e.g. histones and the major nuclear lamina proteins. The results indicate that nascent RNP is more tightly bound to chromosomal DNA than other components that may be involved in nuclear skeletons. This suggest that transcription complexes represent at least one type of anchorage structure of DNA, which is consistent with results indicating that nascent RNA and actively transcribed DNA sequences are preferentially retained in high-salt-treated nuclei.

Animals↗
PubMed1990

Fragmentation of re-formation of mitotic Golgi apparatus detected by a centrifugal method.

The fragmentation/re-formation process of the Golgi apparatus during mitosis was studied by flotation centrifugation in a stepwise sucrose density gradient. The mitotic Golgi fraction was obtained from Chinese hamster ovary cells synchronized with thymidine and nocodazole. The Golgi apparatus detected by a marker enzyme, galactosyltransferase, was separated into two peaks by the flotation centrifugation. The amount of the Golgi recovered at the lower density peak was less in the mitotic cells than in the interphase cells. The separation profile of the mitotic Golgi returned to that of the interphase Golgi by further incubation of the mitotic cells. The re-formation of the fragmented Golgi was inhibited by nocodazole and vinblastine, but not by actinomycin D and cycloheximide.

Animals↗
PubMed1996

Comparison of 2 centrifugation procedures in the formalin-ethyl acetate stool concentration technique for the detection of Cryptosporidium oocysts.

A comparison between the standard formalin-ethyl acetate (FEA) stool concentration procedure (centrifugation at 400 g for 2 min) and a modification proposed to increase the recovery of Cryptosporidium oocysts (500 g for 10 min) was performed on 73 formalin-preserved stool specimens known to be positive for Cryptosporidium. False-negative results were obtained with both methods, but the increase in centrifugation time and force resulted in the detection of a higher number of oocysts and positive samples. The modified procedure had a sensitivity significantly higher than the standard method (99% compared to 86%, P = 0.0045).

Acetates↗
PubMed1990

Effect of centrifugal force and catecholamines on glycosaminoglycans synthesis of vascular smooth muscle cells in culture.

To evaluate the effect of hypertension on glycosaminoglycan (GAG) synthesis, cultured vascular smooth muscle cells (CVSMCs) from the aorta of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were exposed to centrifugal forces and catecholamines. GAG synthesis of CVSMCs was measured by the incorporation of [3H]glucosamine into GAGs which were secreted into the culture medium for 24 h. Basal level of GAG synthesis was much higher in SHR than in WKY, when expressed in terms of DNA contents. When exposed to centrifugal force, CVSMCs from rats of both strains synthesized more GAGs. GAG synthesis was enhanced by both noradrenaline (NA) and adrenaline (Ad) in WKY. The enhanced GAG synthesis in WKY by NA or Ad was prevented by pretreatment with propranolol, but not prazosin. In SHR, NA and Ad did not enhance GAG synthesis at this concentration of catecholamines. However, the effects of propranolol or prazosin on GAG synthesis in SHR, when incubated with either NA or Ad, were compatible with the phenomena observed in WKY. Adding dibutyryl cyclic AMP to the culture medium enhanced GAG synthesis in rats of both strains. These data suggest that not only the mechanical stress of high intra-arterial pressure but also beta receptor stimulation, via increasing cyclic AMP, enhance GAG synthesis of vascular smooth muscle cells in hypertension.

Animals↗
PubMed1981

Separation of guinea pig peripheral blood lymphocytes by discontinuous density gradient centrifugation using Ficoll-metrizoate.

We present a simple method for the recovery of lymphocytes from guinea pig peripheral blood by discontinuous density gradient centrifugation using Ficoll-metrizoate solutions. This technique is formulated specifically for the routine preparation of guinea pig lymphocytes for in vitro cultivation and is capable of recovering 40-60% of available lymphocytes. Whole, heparinized (10-100 U/ml) blood was drawn from strain 2 guinea pigs by cardiac puncture, diluted 1 : 4 with Ca2,Mga free Hanks' balanced salt solution (CMF-HBSS) containing EDTA (5 mM) and gentamicin (50 microgram/ml), and incubated at room temperature for 30-60 min to promote leukocyte disaggregation. Five volumes of diluted blood were layered onto 2 vol of Ficoll-metrizoate adjusted to a density of 1.107 g/ml with sodium metrizoate solution. A band of mononuclear cells (80-90% lymphocytes, 10-20% monocytes, and less than 2% granulocytes) formed at the interface after centrifugation (400 X g, 20-40 min, room temperature). More than 95% of the cells were viable by trypan blue exclusion. Lymphocytes recovered from as little as 3-5 ml whole blood were more sensitive to antigen- or mitogen-activated transformation than leukocyte suspensions obtained by dextran-citrate sedimentation with or without nylon column filtration.

Animals↗
PubMed1981

Assessment of antibody mediated cytolysis of adult cardiocytes isolated by centrifugation in a continuous gradient of Percoll in patients with acute myocarditis.

Principal objections to conventional cytotoxicity assays in cardiac disease with myocytes as target cells are the use of fetal or neonatal myocardium, the cell-membrane of which does not express all antigenic determinants, and the use of trypsin as enzyme for isolation of the cells, since this alters the myolemmal membrane considerably. An improved and rapid procedure for the isolation of intact adult cardiocytes with collaggenase was developed. by means of a performed continuous self-generating silica sol and gradient centrifugation average enrichment of 81% vital myocytes was achieved by a single isopycnic procedure. The yield was improved to 94 +/- 3% vital cells by identical second centrifugation. Cardiocytes isolated by this method were used as target cells in an assay measuring the cytolytic activity of antibodies in the presence of complement: sera of patients suffering from acute viral myocarditis (Coxsackie B- and influenza-virus) with complement fixing antisacrolemmal antibodies (ASA) of the IgG- and IgM-type showed significant cardiocytolysis. ASA are postulated to play a role in the pathogenesis of acute Coxsackie B- and influenza-virus myocarditis.

Acute Disease↗
PubMed1982

A two-step procedure for obtaining normal peripheral blood T-lymphocytes using continuous equilibrium density gradient centrifugation on percoll.

Equilibrium centrifugation of either peripheral blood mononuclear cells or of pure lymphocytes (obtained by carbonyl iron or glass bead adherence removal of monocytes) on a continuous density gradient of Percoll yielded lymphocyte fractions containing between 92 and 99% T lymphocytes as shown by sheep red blood cell rosetting. B lymphocytes with surface immunoglobulin were found in the regions of low density (1.03-1.065 g/ml) and T lymphocytes in the regions of higher density (1.06-1.08 g/ml). TM lymphocytes with their characteristic positive 'dot' pattern of staining for non-specific esterase were also found mainly in regions of high density. It was concluded that Percoll continuous equilibrium density gradient centrifugation can be used to obtain T lymphocytes in high yield, with high viability and without metabolic changes which may occur after contact with sheep red blood cells. The esterase staining suggested that there was also some separation of T lymphocyte subsets.

Cell Separation↗
PubMed1983

Analysis of human peripheral blood lymphocytes isolated by counterflow centrifugation-elutriation.

Human peripheral blood mononuclear cells isolated by Ficoll-Hypaque density gradient centrifugation have been fractionated by counterflow centrifugal elutriation (CCE). Six CCE fractions were obtained and subsequently analyzed as for their content of monocytes, T cells, NK cells and B cells. The various cell types were identified through the expression of specific surface membrane determinants or by cytochemical staining for alpha-naphthyl acid esterase (ANAE). Monocytes were elutriated at the highest counterflow rates whereas the majority of B cells were collected at the lowest counterflow rates. T cells as well as NK cells were mostly concentrated in the intermediate fractions. No differences in the elutriation profile of T cells with the helper-inducer or with the cytotoxic-suppressor surface phenotype were observed. However, the percentages of T cells as determined by surface marker expression decreased with increasing counterflow rates, whereas the percentage of ANAE-positive T cells increased. Yet, T cells recovered at the high counterflow rates had ANAE-reactive organelles larger than those of T cells collected at low counterflow rates. These findings suggest that T cells at different maturational stages could be separated by CCE.

B-Lymphocytes↗
PubMed1983

Cell size monitored counterflow centrifugation of human bone marrow resulting in clonogenic cell fractions substantially depleted of small lymphocytes.

Human bone marrow cells were fractionated by physical methods in order to obtain cell fractions enriched in clonogenic cells and devoid of immunocompetent lymphocytes. The bulk of the erythrocytes was removed by isopycnic gradient centrifugation on Ficoll-Isopaque (d = 1.085 g/ml) and the majority of mature granulocytes on Percoll (d = 1.070 g/ml). The nucleated cells were separated into fractions by counterflow centrifugation. Continuous monitoring of the effluent of the elutriator by a light scatter device improved the reproducibility of the separation profiles. Progenitor cells did not form a single distinct peak and the maximal enrichment factor was 8.5. Lymphocytes were eliminated almost completely from the progenitor cell rich fraction (both CFU-GM and BFU-E). Physical elimination of lymphocytes from human bone marrow may offer an alternative approach to the prevention of graft-versus-host disease in allogeneic bone marrow transplantation.

Antigens, Surface↗
PubMed1984

Enrichment of macrophages in cell suspensions of human intestinal mucosa by elutriation centrifugation.

To obtain macrophage-rich cell suspensions from human intestinal mucosa, lamina propria specimens were dissociated by incubating in EDTA-collagenase-DNAase solutions and further purified by counter-current centrifugation. During enzymatic incubation macrophage dissociation was linear over the first 8-10 h, reaching a maximum concentration of 10% of total cells and then it plateaued. Counter-current centrifugation resulted in a 5-fold enrichment of macrophages to a mean of 50% with an average recovery rate of 84%. Yields exceeded 0.69 X 10(5) macrophages/g mucosa. Greater than 90% of these cells phagocytosed Staphylococcus aureus, and could be maintained in culture for up to 8 days. Electron microscopy showed satisfactory preservation of the ultrastructure of the macrophages, which also seemed functionally intact.

Cell Separation↗