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 1,747 records · Page 97Linked to original sources
PubMed1982

The effects of centrifugation, various synthetic media and temperature on the motility and vitality of human spermatozoa.

The influence of various technical procedures and diluent media upon human spermatozoa has been tested in vitro. The percentage and velocity of motile spermatozoa were measured objectively using laser Doppler velocimetry. Vitality was determined by fluorescent staining. Centrifugation of semen (diluted 1 : 1 with Tyrode) at forces of 500, 800, 1 800 and 2 500 X g resulted in almost complete sedimentation of the spermatozoa. After resuspension in Tyrode's, spermatozoal motility was not changed significantly. The percentages of motile spermatozoa (measured at 37 degrees C) in Tyrode's and B2 medium remained the same as those in seminal plasma throughout an 8-hr period of incubation at 20 degrees C, while those in Locke's and phosphate-buffered saline (PBS) were significantly lower. The instantaneous modal velocities (Vc) of spermatozoa (also measured at 37 degrees C) in Locks's, Tyrode's and B2 were all significantly increased as compared to those observed in seminal plasma during incubation at 20 degrees C; spermatozoa in PBS showed a significantly reduced velocity compared to seminal plasma, but the difference was not significant. However, the rates of decline of the percentage of motile spermatozoa and of their velocities were indistinguishable in all four synthetic media and in the seminal plasma. No significant change in vitality was observed in any medium during the 8-hr incubation period at 20 degrees C. When spermatozoa were incubated at 37 degrees C, rapid declines in both percentage motile and in velocity were observed with incubation periods lasting more than 4-h. Subjecting the spermatozoa to a temperature of 4 degrees C for 1-h did not significantly change either of the motility parameters (measured at 37 degrees C), although prolonged exposure to 4 degrees C did greatly reduce their motility and velocity percentages. The decline in vitality was slow and uniform during incubation at either 4, 20 or 37 degrees C over periods of up to 10-hr, the best survival apparently being obtained at 20 degrees C and the worst at 4 degrees C, although there were no significant temperature differences at any time.

Cell Survival↗
PubMed1969

Equilibrium measurements of embryonic chick cell adhesiveness. I. Shape equilibrium in centrifugal fields.

Sorting-out and other morphogenetic rearrangements of embryonic cells have been attributed to the spontaneous movements of mutually adhesive cells toward configurations of minimal adhesive free energy. Testing this hypothesis requires measuring those particular adhesive properties that would determine such equilibrium configurations. We have devised a modification of the sessile drop method for making the required measurements. This communication is a preliminary report of experiments demonstrating that intercellular adhesiveness is reflected in the equilibrium shapes of centrifuged cell aggregates.

Adhesiveness↗
PubMed1976

Analysis of ion distribution in subcellular myocardial particulates separated by density gradient centrifugation with nonpolar liquid under ion immobilization conditions.

Methods have been developed for the measurement of the Ca and Mg contents of subcellular particulates, separated by nonpolar liquid density gradient ultracentrifugation. After rapid deep freezing and removal of water by freeze-drying the frozen crushed tissue, homogenization and sonification a nonpolar liquid were carried out. Application of this technique has yielded evidence that the relative amounts of Mg and Ca are greatly different in the several subcellular particulates of normal heart muscle separated by density gradient centrifugation. It also appears that the comparative specific activity of Ca, after loading the perfusates with 45Ca, is very different in the several density fractions of the normal heart. The density site of greatest loading with 45Ca is changed in Ca2+-lack arrest and is very greatly altered by the induction of paradoxical Ca contracture. The method of ion-immobilization separation for water-free particulates of different densities in nonpolar liquids appears to be applicable to analysis for various constituents of other biological materials.

Animals↗
PubMed2003

Purification of avian circulating primordial germ cells by nycodenz density gradient centrifugation.

1. Primordial germ cells (PGCs) are the progenitor cells for gametes. In aves, PGCs show a unique migration pathway, that is, they circulate temporarily through the bloodstream during early development. 2. In this study we developed a method to purify circulating primordial germ cells (cPGCs) in quail and chicks by Nycodenz density gradient centrifugation. 3. The process consisted of primary and secondary purification. In primary purification, cPGCs were enriched at the interface of 8 and 12% Nycodenz fractions. In secondary purification, cPGCs were harvested from 8% Nycodenz fraction at a purity of 90% and from 10% Nycodenz fraction at a purity of 70%. The recovery rate of cPGCs was over 70%. 4. This method would facilitate research on cPGCs' culture and the production of transgenic birds using cPGCs.

Animals↗
PubMedDate not supplied

Effect of two-round Ficoll-Hypaque density gradient centrifugation on lymphocyte subsets and natural killer activity of umbilical cord blood mononuclear cells.

Umbilical cord blood (CB) mononuclear cells (MNCs) obtained from Ficoll-Hypaque density gradient centrifugation (FDGC) are frequently contaminated with erythrocytes and nucleated erythroid precursors. The authors investigated the effect of two-round (2-r) FDGC on lymphocyte subsets and natural killer activity of CB and adult peripheral blood (APB) MNCs, in comparison with those obtained from conventional one-round (1-r) separation. The percentage of CD45-expressing CB MNCs was greatly increased after the second density step (p < .01), indicating the efficacy in purification. The percentages of CD3/CD4, CD3/CD8, and CD16/56 double-positive staining CB MNCs were significantly increased after 2-r FDGC, as compared to those after 1-r separation. However, the percentages of CD34+ stem cells and CD19+ B cells were not affected by 2-r FDGC. MNCs obtained from 2-r FDGC had higher natural killer (NK) activity than did MNCs obtained from 1-r separation (p < .01). In contrast, 2-r FDGC did not affect lymphocyte subsets and NK activity of APB MNCs as compared to 1-r FDGC. Thus, 2-r FDGC are recommended to obtain CB MNCs for flow cytometric analysis and NK cytotoxicity assays.

Cell Survival↗
PubMed1991

Comparison of DNA double-strand break rejoining as measured by pulsed field gel electrophoresis, neutral sucrose gradient centrifugation and non-unwinding filter elution in irradiated plateau-phase CHO cells.

The initial (up to 30 min) rate of DNA double-strand break (dsb) rejoining was measured in irradiated plateau-phase CHO cells, in a set of parallel experiments using the same cell suspension, by means of non-unwinding filter elution, neutral sucrose gradient centrifugation, and two pulsed-field gel electrophoresis assays: asymmetric field inversion gel electrophoresis (AFIGE) and clamped homogeneous electric field (CHEF) gel electrophoresis. The rate of DNA dsb rejoining was compared to the rate of rejoining of chromatin breaks measured, also in the same cell population, using the technique of premature chromosome condensation (PCC). Two radiation exposures, 25 Gy and/or 50 Gy, were used and applied to the individual parts of the experiments according to the sensitivity of the assay under investigation. Similar values for the initial rate of DNA dsb rejoining were obtained with all assays used, with t 1/2 ranging between 10 and 12 min after exposure to 25 Gy and between 15 and 20 min after exposure to 50 Gy. The initial rate of rejoining of chromatin breaks was slower than that of DNA dsb and occurred with t 1/2 of 87 min. The results suggest that all major techniques currently used for assaying rejoining of DNA dsb give similar results despite their widely different biophysical basis, and indicate that more information is required before a direct correlation between rejoining of DNA dsb and rejoining of chromatin breaks can be established.

Animals↗
PubMed2006

Establishment of subcellular fractionation techniques to monitor the intracellular fate of polymer therapeutics I. Differential centrifugation fractionation B16F10 cells and use to study the intracellular fate of HPMA copolymer - doxorubicin.

Polymer therapeutics are being designed for lysosomotropic, endosomotropic and transcellular drug delivery. Their appropriate intracellular routing is thus crucial for successful use. For example, polymer-anticancer drug conjugates susceptible to lysosomal enzyme degradation will never deliver their drug payload unless they encounter the appropriate activating enzymes. Many studies use confocal microscopy to monitor intracellular fate, but there is a pressing need for more quantitative methods able to define intracellular compartmentation over time. Only then will it be possible to optimise the next generation of polymer therapeutics for specific applications. The aim of this study was to establish a subcellular fractionation method for B16F10 murine melanoma cells and subsequently to use it to define the intracellular trafficking of N-(2-hydroxyproplylmethacrylamide) (HPMA) copolymer-bound doxorubicin (PK1). Free doxorubicin was used as a reference. The cell cracker method was used to achieve cell breakage and optimised to reproducibly achieve approximately 90% breakage efficiency. This ensured that subsequent subcellular fractionation experiments were representative for the whole cell population. To characterise the subcellular fractions obtained by differential centrifugation, DNA (nuclei), succinate dehydrogenase (mitochondria), N-acetyl-beta-glucosaminidase (lysosomes), alkaline phosphatase (plasma membrane) and lactate dehydrogenase (cytosol) were selected as markers and their assay was carefully validated. The relative specific activity (RSA) of the fractions obtained from B16F10 cells were: nuclei (2.2), mitochondria (4.1), lysosomes (3.7) and cytosol (2.5). When used to study the intracellular distribution at non-toxic concentrations of PK1 and doxorubicin, time-dependent accumulation of PK1 in lysosomes was evident and the expected nuclear localisation of free doxorubicin was seen. Live cell fluorescence microscopy and confocal co-localisation studies gave qualitative corroboration of these results, but by using this method, we were unable to accurately define organelle localisation. In conclusion, the B16F10 subcellular fractionation method developed here provides a useful tool to allow comparison of the intracellular trafficking of other polymer conjugates.

Alkaline Phosphatase↗
PubMed1999

Effect of elution modes on protein separation by cross-axis coil planet centrifuge with two different types of coiled columns.

Effect of elution modes on protein separation was investigated using cross-axis coil planet centrifuge (cross-axis CPC) with two different types of coiled columns, i.e., eccentric coil and toroidal coil assemblies. Myoglobin and lysozyme were separated with an aqueous two-phase solvent system composed of 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate. The substantial effect of elution modes was observed by the toroidal coil, while the negative result was given at the eccentric coil. Using the toroidal coil, higher peak resolution of proteins was attained at the tail to head elution mode. In the outward lower phase mobile elution mode, the satisfactory separation was obtained by both eccentric coil and toroidal coil assemblies. However, in the inward upper phase mobile elution mode, the toroidal coil produced a broad and asymmetric myoglobin peak. The analysis using polyacrylamide gel electrophoresis revealed that the toroidal coil partially separated the components originally present in the myoglobin sample. As the result, a modified equation was devised to express the peak resolution (Rs) using the lysozyme peak. The overall results indicated that the toroidal coil produced better partition efficiency than the eccentric coil under the optimized experimental condition including the direction of the Coriolis force acting on the mobile phase in the toroidal coil.

Centrifugation↗
PubMed2000

The usefulness of blood culture in diagnosing HIV-related systemic mycoses: evaluation of a manual lysis centrifugation method.

The results of 5034 blood cultures, implementing a lysis-centrifugation method with saponin, are summarized in this paper. Three hundred and twenty-two blood samples (6.3%) obtained from a pool of human immunodeficiency virus (HIV)-positive patients yielded fungi. Cryptococcus neoformans was isolated in 199 samples (3.95%), Histoplasma capsulatum in 95 (1.89%). Candida parapsilosis in 12 (0.23%), C. albicans in 7 (0.13%), C. tropicalis in 2, C. krusei in 1, C. guillermondii in 1, and Prototheca wickerhamii in 4 (0.07%). Blood cultures were positive for C. neoformans in 76.23% of patients having a diagnosis of cryptococcosis and in 89.65% of those who had histoplasmosis. The blood culture was the first means of confirming the diagnosis in 23.8% of the patients with cryptococcosis and in 54% with histoplasmosis. In the four patients in whom P. wickerhamii was isolated, a diagnosis of disseminated protothecosis was not achieved by other findings. Catheter infections were responsible for the majority of recovered Candida spp.

AIDS-Related Opportunistic Infections↗
PubMed1999

Effects of process variables on the size, shape, and surface characteristics of microcrystalline cellulose beads prepared in a centrifugal granulator.

Preparation of microcrystalline cellulose (MCC) beads with a laboratory-scale centrifugal granulating apparatus was studied, and the pharmaceutical quality of the beads was characterized with respect to the subsequent drug layering. Five process parameters of potential importance, including rotor rotation speed X1, slit air X2, spray air pressure X3, spray air rate X4, and height of nozzle setting X5, were evaluated using a fractional factorial design (FFD 2(5-2)) as the experimental design. The responses evaluated were expected yield, mean size, size distribution, shape characteristics (including roundness, circularity, elongation, rectangularity, and modelx), and friability. All five process parameters studied were found to have an influence on the selected properties of the beads, but the effects of rotor rotation speed, slit air flow rate, and spray air rate were statistically significant (p < .05). The effect of the rotor rotation speed was found to be the most potent on all the responses studied. The results also show some significant interactions between the parameters tested. The most significant interactions were between rotor rotation speed and slit air, rotor rotation speed and spray air, and slit air and spray air.

Cellulose↗
PubMed1958

Biological activity of fractions obtained by differential centrifugation of the bovine adrenal cortex.

This study is an attempt to correlate adrenocortical hormonal activity with various cell fractions obtained by homogenization and differential centrifugation of bovine adrenocortical tissue. Bioassays involving the decrease in circulating eosinophils and sodium retention were employed for the estimation of the glucocorticoids and mineralocorticoids respectively. The supernatant was the most active fraction in both bioassays, while the microsome and mitochondrial fractions demonstrated varying degrees of activity. The total nitrogen, lipide phosphorus, and cholesterol contents of the fractions were determined. The microsome fraction contained a relatively higher amount of lipide phosphorus and a marked amount of cholesterol.

Adrenal Cortex↗
PubMed1970

Lysosomes in skeletal muscle tissue. Zonal centrifugation evidence for multiple cellular sources.

Postnuclear supernates from homogenates of skeletal muscle from rats subjected to starvation, injections of Triton WR-1339, dextran-500, and dextran + corticosterone were fractionated by means of rate and isopycnic zonal centrifugation in sucrose-0.02 M KCl gradients. Zonal fractions were analyzed for protein, RNA, cytochrome oxidase, and up to six acid hydrolases. The results indicate the presence of two groups of lysosome-like particles. One group contributes approximately 95% of the cathepsin D and acid phosphatase activity and 75% of the acid ribonuclease, beta-glucuronidase, and arylsulfatase activity in muscle. It is characterized by a modal equilibrium density of 1.18 that is decreased by starvation, but is not shifted by dextran-500 or Triton WR-1339. The second group has a higher proportion of acid ribonuclease, beta-glucuronidase, and arylsulftase; the equilibrium density can be shifted by dextran-500 and Triton WR-1339. It is suggested that this group of lysosomes is derived from macrophages and other connective tissue cells, whereas the former group represents lysosome-like particles from muscle cells.

Acid Phosphatase↗
PubMed1974

Analytical study of microsomes and isolated subcellular membranes from rat liver. 3. Subfractionation of the microsomal fraction by isopycnic and differential centrifugation in density gradients.

Rat liver microsomal fractions have been equilibrated in various types of linear density gradients. 15 fractions were collected and assayed for 27 constituents. As a result of this analysis microsomal constituents have been classified, in the order of increasing median density, into four groups labeled a, b, c, and d. Group a includes: monoamine oxidase, galactosyltransferase, 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and cholesterol; group b: NADH cytochrome c reductase, NADPH cytochrome c reductase, aminopyrine demethylase, cytochrome b(5), and cytochrome P 450; group c: glucose 6-phosphatase, nucleoside diphosphatase, esterase, beta-glucuronidase, and glucuronyltransferase; group d: RNA, membrane-bound ribosomes, and some enzymes probably adsorbed on ribosomes: fumarase, aldolase, and glutamine synthetase. Analysis of the microsomal fraction by differential centrifugation in density gradient has further dissociated group a into constituents which sediment more slowly (monoamine oxidase and galactosyltransferase) than those of groups b and c, and 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and the bulk of cholesterol which sediment more rapidly (group a2). The microsomal monoamine oxidase is attributed, at least partially, to detached fragments of external mitochondrial membrane. Galactosyltransferase belongs to the Golgi complex. Group a2 constituents are related to plasma membranes. Constituents of groups b and c and RNA belong to microsomal vesicles derived from the endoplasmic reticulum. These latter exhibit a noticeable biochemical heterogeneity and represent at the most 80% of microsomal protein, the rest being accounted for by particles bearing the constituents of groups a and some contaminating mitochondria, lysosomes, and peroxisomes. Attention is called to the operational meaning of microsomal subfractions and to their cytological complexity.

Aminopyrine N-Demethylase↗
PubMed1980

Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation.

Neutrophil chemotaxis, phagocytosis, and oxygen-dependent microbicidal activity are initiated by interactions of stimuli with the plasma membrane. However, difficulties in neutrophil plasma membrane isolation have precluded studies on the precise structure or function of this cellular component. In this paper, a method is described for the isolation of representative human neutrophil plasma membrane vesicles, using nitrogen cavitation for cell disruption and a combination of differential centrifugation and equilibrium ultracentrifugation in Dextran gradients for membrane fractionation. Multiple biochemical markers and galactose oxidase-tritiated sodium borohydride surface labeling were employed to follow the yield, purity, and distribution of plasma membranes, nuclei, lysosomes, endoplasmic reticulum, mitochondria, and cytosol. According to these markers, neutrophil plasma membranes were exposed to minimal lysosomal hydrolytic enzymes and could be isolated free of other subcellular organelles. In contrast, disruption of neutrophils by mechanical homogenization resulted in > 20% lysosomal rupture and significant plasma membrane proteolysis. Electron microscopy demonstrated that plasma membranes isolated after nitrogen cavitation appeared to be sealed vesicles with striking homogeneity.

Cell Fractionation↗
PubMed1982

Centrifugation shearing exposes filamentous networks in cortical regions of crane-fly spermatocytes.

Spermatocytes of the crane-fly, Nephrotoma suturalis, were attached to electron microscope grids and then sheared by applying centrifugal force. Transmission electron microscopy of exposed regions of the cell cortex revealed networks containing arrays of filamentous structures. Networks were present in sheared spermatocytes at all stages of meiosis. The networks of dividing spermatocytes (meta- through telophase) were denser and appeared to contain more aggregated material then networks of prophase cells. The appearance of networks in spermatocytes resembled actin-containing networks of sheared and detergent-extracted human erythrocytes. Networks treated with myosin subfragment 1 under conditions in which muscle F-actin was clearly decorated were not distinguishable from those of untreated cells. Exposure to deoxyribonuclease-1 caused the disruption of networks in sheared spermatocytes as well as in erythrocytes. The results of deoxyribonuclease experiments are interpreted as an indication that actin is a component of the cell cortex in crane-fly spermatocytes.

Actins↗
PubMed1982

Mitochondrial ribosome assembly in Neurospora. Structural analysis of mature and partially assembled ribosomal subunits by equilibrium centrifugation in CsCl gradients.

In Neurospora, one protein associated with the mitochondrial small ribosomal subunit (S-5, Mr 52,000) is synthesized intramitochondrially and is assumed to be encoded by mtDNA. When mitochondrial protein synthesis is inhibited, either by chloramphenicol or by mutation, cells accumulate incomplete mitochondrial small subunits (CAP-30S and INC-30S particles) that are deficient in S-5 and several other proteins. To gain additional insight into the role of S-5 in mitochondrial ribosome assembly, the structures of Neurospora mitochondrial ribosomal subunits, CAP-30S particles, and INC-30S particles were analyzed by equilibrium centrifugation in CsCl gradients containing different concentrations of Mg+2. The results show (a) that S-5 is tightly associated with small ribosomal subunits, as judged by the fact that it is among the last proteins to be dissociated in CsCl gradients as the Mg+2 concentration is decreased, and (b) that CAP-30S and INC-30S particles, which are deficient in S-5, contain at most 12 proteins that are bound as tightly as in mature small subunits. The CAP-30S particles isolated from sucrose gradients contain a number of proteins that appear to be loosely bound, as judged by dissociation of these proteins in CsCl gradients under conditions in which they remain associated with mature small subunits. The results suggest that S-5 is required for the stable binding of a subset of small subunit ribosomal proteins.

Centrifugation, Isopycnic↗
PubMed1983

Measurement of erythrocyte deformability using a stroboscopic recording centrifuge.

A stroboscopic recording centrifuge technique is described which can measure deformability of red blood cells in terms of the rate of packing at low g. It can be used to measure several whole blood samples simultaneously, with accurate control of the speed of rotation and recording time. Only 0.05 ml of blood is required per measurement, and no prior treatment or preparation of the sample is necessary. The calibration procedures to allow for variations of the haematocrit and plasma fibrinogen level are explained.

Calibration↗
PubMed2003

Dendritic cell culture: a simple closed culture system using ficoll, monocytes, and a table-top centrifuge.

Dendritic cells (DCs) are potent antigen-presenting cells involved in the induction of T cell-mediated immune responses and as such have emerged as important candidates for cellular-based therapies. Critical to safe clinical use is the easy manipulation of DCs and their precursors in a closed system. We have developed a serum-free, closed culture system applying a simple wash-Ficoll centrifugation method to reduce platelet and red blood cell (RBC) contamination. This procedure optimized adherence of monocytes (44 +/- 10.9% recovery, >85% expressed CD14(+)/CD163(+)) for the generation of DCs from mononuclear cell (MNC) apheresis units. Most RBCs and up to 98% of platelets were removed. Following density sedimentation, cell viability remained high (98 +/- 2%) with only minimal loss of monocytes (3 +/- 3%). Importantly, Ficoll-treated monocytes retained their ability to differentiate to mature DCs demonstrated by morphology, phenotype (MHC class II(+), CD1a(+), CD80(+), CD86(+), and CD83(+)), ability to stimulate mixed lymphocyte responses (MLR), present antigen, and produce interleukin-12 (IL-12). Nonadherent CD3(+) (80 +/- 4%) were also isolated for functional assays. Ficoll can be easily incorporated into a simple adherence-based closed system for collection of lymphocytes and adherent monocytes for DC culture. The procedure is relatively fast (effective working time 5-6 h), does not impair monocyte function or induce substantial cell activation, and can be performed economically using equipment found in a typical blood banking environment.

Annexins↗