Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “CENTRIFUGATION”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 847 records · Page 47Linked to original sources
PubMed1996

Spermatozoa selection by the swim-up procedure and two-layer percoll gradient centrifugation.

The efficiency of the swim-up procedure and two-layer Percoll gradient centrifugation in procession of spermatozoa was assessed in ejaculates from 47 infertile men. A significantly higher total number of spermatozoa was harvested from Percoll gradients than from the swim-up procedure, the loss rates in concentration being -13.6 +/- 6.4% and -70.8 +/- 5.8%, respectively (p < 0.0001). Recovery in per cent motility was significantly higher after the Percoll gradient than after the swim-up procedure (34.8 +/- 10.2% versus -10.4 +/- 17.2%, p < 0.05). No significant difference was noted between the mean motility grades of the final solutions obtained by the two methods (2.7 +/- 0.2 and 2.0 +/- 0.4, respectively, p > 0.05). When evaluation was conducted within three initial fresh sample concentration categories such as severe oligospermia (lower than 5 x 10(6)/ml), moderate oligospermia (5 to 10 x 10(6)/ml) and mild oligospermia (higher than 10 x 10(6)/ml), the Percoll technique recovered significantly higher number of spermatozoa than the swim-up procedure through all concentration categories (p < 0.05 for each range). Despite being statistically insignificant, Percoll gradients produced final spermatozoa pools with higher per cent motility and motility quality within all concentration ranges. The results suggest that the Percoll gradient centrifugation should be the preferred selection method regardless of the initial fresh sample concentration.

Cell Separation↗
PubMed1984

Assessment of cell-substrate adhesion by a centrifugal method.

A centrifugal method has been evaluated for measuring the strength of Vero Green Monkey kidney cell adhesion to growth surfaces. The centrifugal force necessary to remove cells gave a quantitative measure of cell adhesion and hence the quality of the growth surface. After being subjected to high gravity forces, both the remaining attached cells and the detached cells were viable, indicating the detachment process did not simply rupture the cell. Electron microscope examination of growth surfaces after cell detachment suggested that remnants related to filopodia remained.

Animals↗
PubMed1986

Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations.

A simple yet effective method (iso-density percoll centrifugation) has been developed for consistently preparing isolated rat liver parenchymal cells with over 98% initial viability. The method has been applied to cells isolated by a variety of collagenase digestion techniques. This procedure involves the low-speed centrifugation (50 X g) of the initial cell suspension through a percoll medium having a density of 1.06 g/ml and results in the separation of single and viable parenchymal cells from cell aggregates, debris, and nonparenchymal cells. The enriched parenchymal cells have been shown to be superior to untreated cells by a number of criteria including: preparation homogeneity, cell morphology, maintenance of cytochrome P-450, hormonal responsiveness (measured by the induction of tyrosine aminotransferase after treatment with glucagon or dexamethasone, or both), plasma membrane integrity (determined by both trypan blue exclusion and leakage of glutamic-oxaloacetic transaminase), and the DNA repair capability after treatment with benzo[a]pyrene or 2-acetylaminofluorene.

2-Acetylaminofluorene↗
PubMed1977

Selection of Ehrlich tumor cells in different phases of the cell cycle by ficoll gradient centrifugation.

Mouse Ehrlich ascites tumor cells were centrifuged at low speed on a linear Ficoll gradient. Cells from different fractions of the gradient were collected separately and analysed by cytological and cytochemical methods (DNA content, mean volume and number of cells). Nearly pure populations of cells in G1 or S were selected. A heterogeneous population containing 70% G2 and mitotic cells was also isolated. No ultrastructural alterations were detected in the cells after centrifugation. Selected G1 cells were cultured in vitro, their kinetic parameters were measured and compared with those of the original population. No difference was observed as far as the duration of the cycle of these cells is concerned.

Animals↗
PubMed1984

Separation of human urinary proximal tubular cells from familial hypercholesterolemic homozygotes by Ficoll gradient centrifugation. Morphological and biochemical characteristics.

The isolation of renal proximal tubular (PT) cells from the fresh urinary sediment ( UrS ) of familial hypercholesterolemic (FH) homozygotes using discontinuous Ficoll gradient centrifugation was studied. For comparative purposes, cultured cells derived from normal human UrS or kidney were also studied. Unfractionated PT cells were nucleated and 90-99% of the cells excluded trypan blue. Both the unfractionated and fractionated cultured normal PT cells contained numerous empty cytoplasmic vesicles. In contrast, similar preparations from the UrS of FH homozygotes contained membrane-enclosed cytoplasmic vesicles that stained with the Papanicolaou (Pap) reagent and were strongly positive with a fluorescein-labeled antibody against lactosylceramide. The PT cells of FH homozygotes contained 2.0 to 2.5 fold higher activity of gamma-glutamyltransferase and alkaline phosphatase, respectively, than the unfractionated UrS cells. We conclude that human PT cells can be separated from other UrS cells by ficoll gradient centrifugation, and that most, if not all of the LacCer present in the UrS of FH homozygotes is associated with the PT cells. Purified PT cells should provide a useful model to test the biochemical mechanisms of LacCer accumulation in FH.

Adolescent↗
PubMed1990

Differential effect of pH on the density and volume of rat reticulocytes and erythrocytes. Relevance to their fractionation by centrifugation.

Rats were injected with 59Fe-ferrous citrate and bled thereafter at different times (16 h to 49 d). This gave rise to red cell populations in which cells corresponding in age to the time elapsed between injection and bleeding were labeled. The anticoagulant used was either acid-citrate-dextrose (ACD) with a pH adjusted to 7.3 or ACD (pH 5.1). Final pH of the collected blood was about 7.2-7.4 in the former case and 6.4-6.7 in the latter. Red cells were then centrifuged (5) and approximately 7-10% of the packed cells from the top and 7-10% from the bottom of the cell column collected. When reticulocytes are the predominant labeled red cell population, as in blood obtained for about 24 h after isotope injection, a fractionation of these cells and mature erythrocytes is in evidence only when blood is collected at the higher pH. Thus, at pH 7.2-7.4 ratios of specific radioactivities of cells in top fraction/cells in an unfractionated sample are about 3, whereas at pH 6.4-6.7, the analogous ratios are 1 or less. These differences in specific activity ratios, as a function of pH at collection, virtually disappear after about 4 d following isotope injection. The lower pH is known to increase the volume and decrease the density of mature red blood cells. The marked effect of pH on cellular fractionation could be correlated with the smaller change in rat reticulocyte density and volume in acid medium. At pH 6.4-6.7, the densities of mature erythrocytes and reticulocytes are so close that their physical separation by centrifugation is not feasible.

Animals↗
PubMed2004

Moving objects in a rotating environment: rapid prediction of Coriolis and centrifugal force perturbations.

Grip force adaptation to Coriolis and centrifugal force perturbations was tested in healthy subjects. Eight subjects were seated in a rotating chamber in a rotating axis position. They each grasped an instrumented object resting on the thumb, which was stabilized by the other fingers from above. Subjects performed horizontal point-to-point movements with the grasped object away and towards the trunk. These movements were directed in a nonparallel fashion towards the axis of rotation prior (40 pre-rotational movements), during (80 per-rotational movements) and following (40 post-rotational movements) clockwise body rotation. During pre- and post-rotational movements two load force peaks of similar magnitude occurred during the acceleratory and deceleratory phases of the movements. Accordingly, a Coriolis force, which was orthogonal and proportional to the linear velocity of the moving arm, as well as a centrifugal force proportional to the system's squared angular velocity and movement amplitude developed during per-rotational movements. The load perturbations altered the load force profile in a characteristic way. The first 10 per-rotational movement sequence revealed that there was a less precise coupling between grip and load force magnitudes and a reduced temporo-spatial co-ordination between grip and load force profiles. With increasing number of per-rotational movements, there was significant improvement in the temporo-spatial co-ordination and in the coupling in force magnitude between grip and load force profiles, indicating an ongoing adaptation process. The coupling between grip and load forces proved to be similarly precise for the last 10 per-rotational movements and for pre-rotational movements, suggesting complete adaptation. Significant effects were observed for the first post rotational movements following adaptation to the per-rotational load characteristics both for the temporal co-ordination between grip and load forces and for the coupling in force magnitudes. However, the last 10 post-rotational movements proved to be similarly precise with comparison to pre-rotational performance in terms of grip force regulation with movement-induced loads. The results are discussed within the context of the CNS ability to use internal models when planning and processing anticipatory grip force adjustments during manipulative tasks.

Acoustic Stimulation↗
PubMed2002

Separation of neonatal rat ventricular myocytes and non-myocytes by centrifugal elutriation.

The preparation of pure cardiac myocyte cultures from neonatal rats is hampered by the presence of non-myocytes, which can proliferate during culturing, thereby causing a progressive decrease in the proportion of myocytes. In order to obtain myocyte cell suspensions of high purity, a method based on centrifugal elutriation was developed. Cardiac cells, isolated from neonatal rat heart ventricles, were subjected to elutriation using flow rates that increased step-wise from 20 to 80 ml/min. The cell fraction obtained at 80 ml/min consisted of 68-90% myocytes. Still, upon culturing, the remaining non-myocytes proliferate, causing the proportion of myocytes to decrease to 60 +/- 2% at day 5. A second elutriation protocol was developed in which myocytes and non-myocytes were separated after a period of co-culturing for 4-5 days. By this approach a fibroblast-rich cell fraction (87 +/- 5%) and a myocyte-rich cell fraction (82 +/- 6%) were obtained. In conclusion, centrifugal elutriation creates the opportunity to separate neonatal rat myocytes from non-myocytes, either freshly isolated or after a period of culturing. Particularly, cell separation after a period of culturing ventricular cells offers an advantage to analyse the experimental effects on myocytes and non-myocytes separately.

Animals↗
PubMed2004

Clinical results of extracorporeal membrane oxygenation (ECMO) support for acute respiratory failure: a comparison of a centrifugal pump ECMO with a roller pump ECMO.

PURPOSE: The purpose of this study was to compare the clinical results of extracorporeal membrane oxygenation (ECMO) using a centrifugal pump (CP group) and that using a roller pump (RP group) for the treatment of acute respiratory failure (ARF). METHODS: From November 1990 to July 2001, the ECMO system was introduced for the treatment of 15 patients with ARF; 10 cases of pneumonia or adult respiratory distress syndrome (ARDS), 4 cases of pulmonary hemorrhage, and 1 case of hypoxemia following cardiac surgery. Five patients were included in the RP group, and 10 were included in the CP group. RESULTS: The mean PaO2/FIO2 ratio, PaCO2 prior to ECMO induction, and the mean duration of ECMO support were 59.8, 38.8 mmHg, and 125 h, respectively, in the RP group, and 65.6, 82.0 mmHg, and 107 h, respectively, in the CP group. The mean PaCO2 value was significantly (P < 0.05) higher in the CP group than in the RP group due to the ventilation with "permissive hypercapnia." In the RP group, all patients died of either complications or recurrence due to ARF. In the CP group, 3 patients including 2 with pulmonary hemorrhaging were discharged. CONCLUSIONS: Although the centrifugal pump ECMO improved the clinical results of ARF, the overall prognosis nevertheless depended on the original disease.

Acute Disease↗
PubMed1978

Interaction of antibody with antigen immobilized on polystyrene latex beads: characterization by density gradient centrifugation.

Isopycnic banding by density gradient centrifugation was used to measure density changes in complexes formed by the interaction between antigen and antibody immobilized on polystyrene latex beads (diameter, 0.109 +/- 0.0025 micron). Measurements of density changes allowed calculation of the interacting masses under the given experimental conditions. Interaction equilibrium constants and free energy change for two sets of reactions, bovine IgG and anti-bovine IgG (rabbit) IgG and rabbit IgG and anti-rabbit IgG (goat) IgG systems, were calculated from isopycnic banding density gradient centrifugation runs. The procedure demonstrates a new method of obtaining quantitative information on antigen-antibody interactions.

Animals↗
PubMed1986

Technique and apparatus for automated fractionation of the contents of small centrifuge tubes: application to analytical ultracentrifugation.

An automated method is described for dividing the contents of small cylindrical centrifuge tubes into fractions deriving from laminae of solution as thin as 0.1 mm in the direction of the cylindrical axis. Experimental data are presented to demonstrate that fractions as small as 1 microliter may be prepared with a standard deviation of less than 3% in volume delivery and that negligible mixing occurs between the contents of adjacent fractions during the fractionation procedure. The method has been used to quantitate the gradients of a variety of radiolabeled proteins formed in sedimentation velocity and sedimentation equilibrium experiments. Sedimentation coefficients and molecular weights calculated from the gradients obtained agree well with literature values and with values obtained by optically scanning the centrifuge tubes (A.K. Attri and A.P. Minton, 1983, Anal. Biochem. 133, 142-152; 1984, Anal. Biochem. 136, 407-415). The present technique combines a spatial resolution equal to that of optical methods of gradient measurement with a sensitivity which may be several orders of magnitude greater, depending upon the specific activity of labeled solute.

Animals↗
PubMed1990

Adaptation of an enzymatic cycling assay for NADP(H) measurement to the COBAS-FARA centrifugal analyzer.

NADP(H) measurements by enzymatic amplification are described in which the interface step between cycling (glucose-6-phosphate and glutamic dehydrogenases) and indicator (6-phosphogluconic dehydrogenase) enzymes has been reconfigured, permitting the entire operation to run as a continuous assay on a centrifugal fast analyzer. This is accomplished by using the sequential load feature of the analyzer and incorporating either sodium dodecyl sulfate (SDS) or SDS and hydrogen peroxide as kill reagents to replace the thermal step (destruction of cycle enzymes by boiling). The ability of SDS to render a cycle inoperative during the run time of the indicator enzyme depends on the inherent resistivity and absolute amount of its enzyme proteins to this surfactant. Criteria used to judge the efficacy of a potential kill reagent are based on the sample blank time-response curve and the cycle product recovery by the indicator enzyme. Various other enzyme cycling systems which can be fitted to the centrifugal fast analyzer are highlighted.

Centrifugation↗
PubMed1990

Isolation of cell cycle fractions by counterflow centrifugal elutriation.

Counterflow centrifugal elutriation (CCE) has been used to fractionate cell populations on the basis of sedimentation properties, with minimal perturbation of metabolic function. Therefore, it is an ideal method for the isolation of cell cycle phase specific populations. We present modifications of the standard Beckman centrifugal elutriation system which permit standardization of the elutriation procedure and eliminate inter-run variability. We provide elutriation parameters for the cell cycle fractionation of a variety of cultured cell lines and suggest ways to improve the quality of the cell separations. In addition, we describe protocols for the fractionation of up to 3.50 X 10(8) cells in the small (JE-6B) Beckman elutriation system. This represents a four- to eight-fold increase in cell numbers over current cell fractionation procedures. Cell cycle populations containing greater than 95% G1, greater than 80% S, and greater than 70% G2/M were consistently obtained using these protocols. Finally, we analyzed phase-enriched fractions from several cultured cell lines for the cell cycle regulation of the enzyme thymidine kinase. The data confirm previous findings that CCE is an excellent means of obtaining physiologically unperturbed cell cycle phase specific fractions.

Animals↗
PubMed1992

RNA isolation from cartilage using density gradient centrifugation in cesium trifluoroacetate: an RNA preparation technique effective in the presence of high proteoglycan content.

An efficient method for the isolation of RNA from cartilage is described. The difficulties in obtaining RNA from cartilage, a tissue of low cell density and high proteoglycan content, were overcome by making several modifications to the guanidine thiocyanate/cesium chloride method of RNA extraction. Cartilage tissue is frozen, crushed, and homogenized in a 4 M guanidine thiocyanate lysis buffer. The RNA is then pelleted by ultracentrifugation through a cesium trifluoroacetate density gradient. The use of cesium trifluoroacetate, rather than cesium chloride, for density gradient centrifugation improves both the yield and purity of total RNA isolated from cartilage. The ultracentrifugation has been adapted to the Beckman TL100 tabletop centrifuge and is complete in 3 h. This fast, simple method produces high quality RNA, suitable for use in RNase protection assays, polymerase chain reaction analysis, and Northern analysis. This purification procedure may be applicable to other sources, from which RNA isolation is complicated by the presence of abundant cell wall or matrix components.

Animals↗
PubMed1979

Isolation of neuronal plasma membranes from the crayfish Procamburus clarkii, with an aqueous two phase polymer system followed by sucrose density gradient centrifugation.

An aqueous two phase polymer system (Dextran-polyethyleneglycol system was developed for isolation of plasma membrane fraction from nerves of the crayfish, Procamburus clarkii. The polymer system effectively reduced both mitochondrial and endoplasmic reticulum marker enzyme activity from a crude membrane fraction. The similar enrichment of (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) was shown by the polymer system as well as by the sucrose density gradient centrifugation. The purified plasma membrane fraction (PM) was obtained using the polymer system followed by sucrose density gradient centrifugation. The PM fraction had a high specific activity of (Na + K+)-ATPase of up to 17 times that in the homogenate, with smaller contamination by mitochondria and endoplasmic reticulum enzyme activities than any other membrane fraction. Electron micrographs of the PM fraction also supported the above evidences. The protein recovered from the PM fraction amounted to 1.1% of the total protein in the homogenate. The specific activity of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) in the membrane fractions was less increased than that of (Na+ + K+)-ATPase. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis suggested that polypeptide chains of estimated molecular weight 115,000 and 31,000 were enriched in the plasma membranes of the crayfish nerves.

Animals↗
PubMed1986

Isolation of renal brush-border membrane vesicles by a low-speed centrifugation; effect of sex hormones on Na+-H+ exchange in rat and mouse kidney.

Na+-H+ exchange in rat and mouse renal brush-border membrane vesicles was studied by fluorescence quenching of the delta pH indicator, acridine orange. Brush-border membrane vesicles were isolated by a modified Mg/EGTA-precipitation method at low speed centrifugation (8000 X g). The enzymatic characteristics of these membrane vesicles were similar to those obtained by the original high-speed centrifugation method (Biber et al. (1981) Biochim. Biophys. Acta, 647, 169-176). The rates of Na+-H+ exchange in renal brush-border membrane vesicles from male and female rats were similar. Neither ovariectomy nor treatment of ovariectomized rats with estradiol or testosterone changed the activity of Na+-H+ exchanger. The rates of Na+-H+ exchange in the mouse were smaller than in the rat indicating the existence of species differences. Na+-H+ exchange in mouse renal brush-border membranes exhibit strong sex differences, the rates in the male being higher than in the female. Castration of male mice led to a decrease in Na+-H+ exchange to values found in females. Treatment of castrated mice with estradiol had no effect. In contrast, treatment with testosterone increased the rate of the exchanger by more than 100%. The effect of testosterone was restricted to the Vmax of the Na+-H+ exchanger, whereas the apparent Km for Na+ remained unchanged. Na+-dependent D-glucose transport in mouse renal luminal membranes exhibited also sex differences due to the potent stimulatory effect of testosterone. Therefore, Na+-H+ exchange and Na+-dependent D-glucose transport in the mouse kidney are under control of androgen hormones. This effect could be in close connection with the wellknown renotropic action of androgens in the mouse.

Animals↗
PubMed1987

Isolation of apical plasma membrane in rabbit gallbladder epithelium by Percoll density gradient centrifugation.

The apical membranes of rabbit gallbladder epithelial cells were isolated by treating the homogenate with Ca2+ or Mg2+ and centrifuging the suspension in Percoll gradient. In this way brush-border membranes were obtained with enrichment factors ranging between 10 and 20 and yields of 15-30%. A second method is described with which membranes were isolated, without any preliminary treatment, first by differential centrifugation, then with Percoll gradient; the final membrane enrichment was over 15, however the yield was very low (3%). Many possible enzymatic markers of the apical plasma membrane were investigated: L-gamma-glutamyltransferase, alkaline phosphatase, leucine aminopeptidase, sucrase. The first appears to be that of choice. Apical membrane fraction could be also evidenced by autofluorescence or by labeling with Lotus tetragonolobus lectin. Preliminary experiments showed that apical plasma membranes isolated in this way form vesicles.

Animals↗
PubMed1977

Purification and characteristics of (Na+, K+)-ATPase from canine kidney by zonal centrifugation in sucrose density gradient.

Microsomes were prepared from the outer medulla of canine kidney. Partially purified preparation of (Na+, K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) was obtained by solubilization of microsomes with sodium deoxycholate, and by precipitation with dilution. The deoxycholate-enzyme thus obtained was further purified by incubation with sodium dodecyl sulphate in the presence of ATP followed by a single zonal centrifugation in a sucrose density gradient by the method of Jørgensen [(1974) Biochim. Biophys. Acta 356, 36-52]. The (Na+, K+)-ATPase was purified to a specific activity of 1600--1800 micronmol Pi - h-1 - mg-1 protein. The yield was 20 mg per single centrifugation with a zonal rotor. Electron microscopy showed that the sectioned pellet of the purified enzyme contained exclusively membranous fragments in contrast with membranous vesicles of starting microsomes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that almost all proteins were accounted for by two polypeptides with molecular weights of 105 000 and 58 000, and that the mass ratio of the large to the small polypeptide was 82 : 18.

Adenosine Triphosphatases↗