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 1,675 records · Page 93Linked to original sources
PubMed1999

Infectivity of feline enteroepithelial stages of Toxoplasma gondii isolated by Percoll-density gradient centrifugation.

The infectivity of the feline enteroepithelial stages of Toxoplasma gondii isolated by Percoll-density gradient centrifugation was examined by the trypan blue dye exclusion method by assaying their penetration into feline fibroblast cells in vitro and by inoculation of the intestinal mucosa of cats. A large population of the parasites showed trypan blue dye exclusion activity. When feline fibroblast cells were inoculated with feline enteroepithelial stage parasites, no intracellular parasites were found 18 h post-inoculation. Kittens inoculated intraduodenally with 2 x 10(6) feline enteroepithelial stage parasites shed oocysts between 2 and 8 days post-inoculation. These results indicate that the isolated feline enteroepithelial stage parasites display infectivity towards enterocytes of cats and are capable of gametogenesis.

Animals↗
PubMed2000

Transfection using synthetic peptides: comparison of three DNA-compacting peptides and effect of centrifugation.

Positively charged peptides have been shown to allow efficient transfection in vitro, especially when mixed with lipids. We have compared the ability of three positively charged peptides both to compact DNA and to increase the transfection efficiency of the cationic lipid DOTAP. The peptides are: a polymer of 17 lysines (pK17), YKAWK8WK (peptide K8) and SPKRSPKRSPKR (peptide P2). Peptides pK17 and K8 compact DNA efficiently in a gel retardation assay and protect DNA efficiently against DNase I degradation. Peptide P2, on the other hand, interacts weakly with DNA and provides poor protection. In order to compare their transfection efficiency, the three peptides were mixed with DNA (plasmid pEGFP-N1) at different charge ratios (+/-) and DOTAP (at a charge ratio of 2). The transfection efficiency was measured by FACS analysis at different times post-transfection. With NIH-3T3 cells, peptide P2 provides the highest transfection efficiency (about 40%), when compared with peptides pK17 (29%) and K8 (31%) and DOTAP alone (21%) under optimal conditions. Finally, we showed that centrifugation of the complexes onto the cells increased the transfection efficiency by a factor 1.5 to 2 with the various cell lines tested (ECV, primary human keratinocyte, CFT-2, NT-1).

3T3 Cells↗
PubMed1984

Liquid-liquid extraction of membranes from calf brain using conventional and centrifugal counter-current distribution techniques.

Neural membranes isolated from calf brain have been partitioned in aqueous two-phase systems containing dextran and polyethyleneglycol. When the partition was repeated several times, using counter-current distribution technique, the distribution of the membranes between the upper phase and the interface changed in a non-ideal manner and in favour of the interface. By using a centrifugal counter-current distribution device the time for the experiment could be reduced by a factor of 7-8 and the distribution was similar to what could be expected for ideally behaving membranes. The time-dependent change of the membranes is discussed in terms of aggregation and lateral membrane perturbations. Despite this effect a certain fractionation has been achieved as deduced from analysis of cholesterol content, opiate receptor activity and acetylcholinesterase activity along the counter-current distribution row of fractions. Compared to the starting material these activities were enriched some two-fold in certain fractions.

Animals↗
PubMed1998

Effect of centrifugation on separation by aqueous two-phase partition of an early and late endosome model using inside-out plasma membrane vesicles from plants.

Inside-out vesicles of plasma membranes prepared from a plant source were used as models to investigate effects of centrifugal forces on separations of early and late endosome populations by aqueous two-phase partition. Endosome subpopulations were resolved readily by preparative free-flow electrophoresis where acidification of the interiors of late endosomes occurred upon addition of ATP to activate a proton translocating ATPase. The resultant increased diffusion potential provided for a surface difference between late and early endosomes to permit electrophoretic separation. With the plant membranes, unincubated inside-out plasma membrane vesicles modeled early endosomes, whereas inside-out vesicles incubated with 1 mM ATP modeled late endosomes. A latent, 2,4-dichlorophenoxyacetic acid (2,4-D)-(auxin)-stimulated NADH:protein disulfide reductase measured spectrophotometrically was used as an enzymatic marker for both populations of inside-out vesicles. Phase partition behavior of each population was quantitated using total protein as the parameter.

Adenosine Triphosphate↗
PubMedDate not supplied

Surface markers and biological functions of PMNC fractionated by countercurrent centrifugal elutriation.

Peripheral blood mononuclear cells (PMNC) were separated by countercurrent centrifugal elutriation (CCE) in PBS-EDTA medium into 13 fractions. Properties associated with B cells were restricted mainly to the first fractions (F2 and F4) while those associated with T cells were confined to fractions F4, F6 and F8. The final fraction (FF) contained more than 90% monocytes while fraction F10 contained 40% monocytes and 60% null cells, and F12 contained 50% monocytes and 50% null cells. Natural killer (NK) cell activity against K-562, a human erythroleukaemia cell line, and against an adherent undifferentiated sarcoma target, was distributed between fractions F6 and F8. Using electron and scanning microscopy and a new single cell liquid cytotoxic assay, we studied membrane interaction and binding of the CCE fractionated cells with the 2 targets. Effector/target cell conjugates revealed interdigitations in the area of cell contact but no membrane fusion, although certain effector cells had a distinct large granulocytic lymphocyte morphology. Furthermore, different cell types from different fractions were able to bind to the targets although the killing was associated with certain fractions only. When purified monocytes from fraction FF were added to F6 and F8, NK activity against both targets was depressed. This inhibition was not reversed by indomethacin, and binding of the targets to the effector cells was unaffected. CCE is a powerful technique which allows the fractionation of PMNC without altering their biological functions, contrary to what is seen with nylon wool or dextran fractionation. In this report, the activity associated with CCE fractions is studied and discussed.

Antibody-Producing Cells↗
PubMed1992

Characterization of YB2/0 cell line by counterflow centrifugation elutriation.

The non-secreting rat myeloma cell line YB2/0 could be separated into different cell fractions by counterflow centrifugal elutriation. The obtained fractions are analyzed by morphology studies, morphometrics, clonogenic assays and flow cytometry. The methodology is extensively described. A separation of different cell fractions according to cell cycle stages was achieved. This implies further application possibilities for clinical use like the in vitro fractionation of autologous bone marrow prior to transplantation in patients with multiple myeloma.

Animals↗
PubMed2004

Agrochemical characterisation of "alperujo", a solid by-product of the two-phase centrifugation method for olive oil extraction.

Introduction of the two-phase centrifugation system for olive oil extraction during the early nineties in Spain has led to the generation of approximately four million tons per year of a solid olive-mill by-product called "alperujo" (AL). Agrochemical characterisation showed that AL has a high moisture content, slightly acidic pH values and a very high content of organic matter, mainly composed by lignin, hemicellulose and cellulose. It also has a considerable proportion of fats, proteins, water-soluble carbohydrates and a small but active fraction of hydrosoluble phenolic substances. Amongst plant nutrients, AL is rich in potassium, less so in (mainly organic) nitrogen and poor in phosphorus and micronutrients. Some of those properties are not compatible with agricultural requirements, for which reason composting may be considered a suitable alternative for its disposal. AL needs to be characterised before composting, and so an attempt was made to correlate the easily determined ash content with other parameters; the regression models thus obtained are discussed.

Agriculture↗
PubMed1991

[Preparation of leukocyte-depleted human platelet concentrates by centrifugation and filtration of a pool of sterilely connected buffy-coats].

We describe a new method for the preparation of standardised therapeutic doses of leukocyte depleted platelets. The first step is to remove the buffy-coat from whole blood units drawn on triple Siamese ACD/SAGM bags (Maco-Pharma) by means of a Compomat (NPBI). The second step is to connect (SCD Haemonetics) six buffy-coats and one plasma to a special kit (Maco-Pharma) including a PALL PL 100 filter; after centrifugation, the supernatant platelet concentrate is extracted, filtered and recovered in a 2 litre TOTM PVC bag. The volume, the number of platelets and leukocytes of these pools are measured. A comparison of these parameters is made with therapeutic doses prepared in the same way without filtration. Besides, pH measurements up to the 6th day of storage and bacteriological checks are carried out. The results show: no platelet loss related to filtration; a synergy between the preparation process out of buffy-coats and the filtration: so each dose contains less than 10(6) leukocytes; a good pH level allowing the storage for five days as it is associated to the bacteriological safety of the functionally closed system. This technique makes it possible to transfuse only leukocyte depleted platelet concentrates. In addition, it offers new prospects for standardisation and quality improvement.

Blood Transfusion↗
PubMed1999

Proteasome activator subunit PA28 alpha and related Ki antigen (PA28 gamma) are absent from the nuclear fraction purified by sucrose gradient centrifugation.

The aim of the present work was to attempt to partially purify PA28 (REG) alpha and gamma (Ki antigen) in the nuclear fraction from NT2/D1 cells. Nuclei were isolated by the hypertonic sucrose gradient centrifugation method and fractionated into membrane/nucleoplasmic and chromatin/nucleolar fractions. Western blotting with anti-histone and anti-beta-tubulin monoclonal antibodies confirmed the accuracy of the procedure. Proteasomes were present mainly in the cytoplasm but also in the nuclei. Disruption of the nuclear envelope released the proteasomes implying a loose or no binding with the chromatin. PA28 alpha and gamma were detected mainly in the cytosol and to a lesser extent in the crude nuclear pellet, however the purified nuclei were devoid of PA28 alpha and gamma. This indicates, that only a small fraction of the PA28 activator is present in the nuclei as detected by immunofluorescence or/and it is easily removed during nuclear purification.

Antigens, Surface↗
PubMed1984

The large-scale preparation of purified sporozoites of Eimeria spp. by metrizamide density-gradient centrifugation.

A method is described for the reproducible purification of greater than 1.0 X 10(8) sporozoites of Eimeria spp. by centrifugation on metrizamide density-gradients. The mean contamination of the purified sporozoite fraction by sporocysts and sporocyst debris was less than 6% and the recovered sporozoites were fully viable, infective and ultrastructurally intact.

Centrifugation, Density Gradient↗
PubMed2001

Fractionation of moderate molecular weight polysiloxanes by centrifugal TLC.

Fractionation of polydispersed polysiloxanes (1-2 g) into narrow molecular weight fractions has been achieved by a rapid (30-60 min) convenient process, using the Cyclograph Centrifugal Chromatography System. These narrow molecular weight poly(dimethylsiloxane) fractions can be used as secondary standards for GPC.

Centrifugation↗
PubMed2004

Hybridization enhancement using microfluidic planetary centrifugal mixing.

DNA microarrays produce their greatest sensitivities when hybridized using concentrated samples and effective mixing; however, these goals have proved elusive to combine. If samples are diluted enough to fill larger chambers, then mixing works well using either pumping or gravity with rotation, although sensitivities will suffer. Various techniques for mixing concentrated samples in small thin chambers have been proposed; however, they often leave streaks or scars, and their reusable components require careful cleaning. Here we introduce a versatile new microfluidics platform, a two-axis centrifuge whose fluidic chambers rotate in a planetary relationship to a radial gravitational field. This paradigm readily overcomes surface and viscous forces even in chambers only 50 microm thin. Thin chambers obviate the need for sample dilution and increase sensitivities and dynamic ranges 10-fold. In comparisons against conventional mixing using the same 10 microg of starting total RNA on 22 000-probe arrays, 10 000 more usable signals rose above the noise. In other experiments, planetary mixing was able to produce comparable results while using only one-tenth the starting sample. The benefits of planetary mixing include sample conservation, shorter hybridizations, less reliance on amplification, and the ability to quantify many gene signals otherwise obscured by noise.

Animals↗
PubMed2006

Centrifuge microextraction coupled with on-line back-extraction field-amplified sample injection method for the determination of trace ephedrine derivatives in the urine and serum.

Although sample stacking has enjoyed some degree of success in electrophoretic separation techniques, there is still a major problem with complex matrix sample as it suffers tremendously from sample matrix effects. A novel method that combines two concentration techniques, centrifuge microextraction (CME) and on-line back-extraction field-amplified sample injection (OLBE-FASI), is used to determine trace ephedrine derivatives in urine and serum by capillary zone electrophoresis. The CME, integrating the sample cleanup and preconcentration into a single step, is a promising sample preparation method for biological samples. The CME technique provided 9-14-fold enrichment within 10 min. The OLBE-FASI eliminated the need to perform solvent exchange and provided a further concentration of the analytes. Using CME coupled with OLBE-FASI, over a 3800-fold increase in sensitivity could be obtained as compared with the normal hydrodynamic injection without sample stacking. For a 1-mL urine sample, the linear range was 5/10-200 ng/mL with the square of the correlation coefficients (r(2)) ranging from 0.9988 to 0.9994. Detection limits were from 0.15 to 0.25 ng/mL using a photodiode array UV detection at wavelength 192 nm. The possibility of this method to determine ephedrine derivatives in 20-muL serum samples was also demonstrated.

Centrifugation↗
PubMed1985

Lipid solvation of the aqueous form of the myelin proteolipid apoprotein: evidence and characterization of two lipid populations by fluorescence polarization, differential calorimetry, and sucrose gradient centrifugation.

The interaction between dipalmitoylphosphatidylcholine (DPPC) and the aqueous form of the myelin proteolipid apoprotein (PLA) has been investigated. Lyophilization was found to be an efficient and nonperturbing method for membrane reconstitution. Mixtures of different lipid/protein ratios were analyzed by means of differential calorimetry, fluorescence polarization, and sucrose gradient centrifugation. The presence of two coexisting lipid populations, termed "bulk" and "interacting" lipids, was demonstrated by these three techniques. By differential calorimetry, 23 DPPC molecules per molecule of protein (30 kDa) were shown to be excluded from the lipid phase transition. By fluorescence polarization, we detected above the phase-transition temperature a large perturbation of the lipid acyl chain dynamics induced by the aqueous form of PLA. Increasing the protein content above 35% by weight within the recombinants caused drastic changes in both delta H values and the fluorescence anisotropy parameter, which could stem from protein aggregation.

Animals↗
PubMedDate not supplied

Isolation of human blood cells, platelets, and plasma proteins by centrifugal SPLITT fractionation.

Centrifugal SPLITT fractionation, a technique designed for the continuous high-resolution separation of colloids and low-density particles, is applied here to fresh human blood, producing six purified fractions consisting of proteins and lipoproteins, platelets, red blood cells, lymphocytes, monocytes, and neutrophils. Production of the six fractions requires five steps, each yielding two fractions. These five steps are carried out in sequence using a single apparatus, with conditions varying from step to step in accordance with theoretical guidelines in order to achieve the desired cut points. In the first step, a stream of diluted blood is separated into one fraction consisting of platelets and plasma and another containing blood cells. The throughput of diluted blood is 162 mL/h and that of whole blood is about 2 mL/h or approximately 10(10) cells/h; guidelines are given for significantly increasing throughput. The purity of the blood cell fractions ranged from 92 to 98%, and the viability fell in the range 97-99%.

Blood Cells↗
PubMedDate not supplied

Scale-up of centrifugal impeller bioreactor for hyperproduction of ginseng saponin and polysaccharide by high-density cultivation of panax notoginseng cells.

Scale-up of a novel centrifugal impeller bioreactor (CIB) was demonstrated for production of valuable plant-specific secondary metabolites by high-density cell cultures. Initial kLa was identified to be a key factor affecting cell growth and production of ginseng saponin and polysaccharide by high-density cultivation of Panax notoginseng cells in a 3-L CIB. A high level of ginseng saponin and polysaccharide production was obtained at an initial kLa value of 30.2 h(-1). A maximum dry cell weight (DW) and production titer of ginseng saponin and polysaccharide reached 22.0 +/- 0.3, 1.5 +/- 0.1, and 2.7 +/- 0.2 g/L on day 15 with their corresponding productivity of 1140 +/- 42, 81 +/- 8, and 150 +/- 17 mg/(L.d), respectively. Based on initial kLa level, the CIB high-cell-density cultivation process was successfully scaled up from 3 L to 30 L. A maximum DW and production titer of ginseng saponin and polysaccharide in a 30-L CIB reached 25.5 +/- 0.5, 1.7 +/- 0.1, and 2.9 +/- 0.1 g/L (on day 15) at an initial kLa value of 28.7 h(-1), respectively, and their corresponding productivity was 1340 +/- 56, 91 +/- 9, and 164 +/- 15 mg/(L.d). Furthermore, by adopting a fed-batch cultivation strategy, a maximum DW and concentrations of total saponin and polysaccharide in the 30-L CIB were enhanced to 30.3 +/- 1.0, 2.1 +/- 0.1, and 3.5 +/- 0.2 g/L with their corresponding productivity of 1467 +/- 87, 102 +/- 13, and 179 +/- 18 mg/(L.d), respectively. The work suggests that the CIB may have great potential in large-scale high-density plant cell cultures for efficient production of useful secondary metabolites.

Bioreactors↗
PubMedDate not supplied

Preparative fractionation of protein, RNA, and plasmid DNA using centrifugal precipitation chromatography with tubular dialysis membrane inside a convoluted tubing as separation channel.

Fractionation of clarified E. coli lysate components in bench-scale and preparative-scale centrifugal precipitation chromatography (CPC), using a solution of cationic surfactant cetyltrimethylammonium bromide (CTAB) containing 0.5 M NaCl as precipitant, are compared here. Step gradient of CTAB from 0.50% to 0.16% (w/v) gave a successful fractionation in bench-scale CPC; however, a linear gradient of lower CTAB concentration, 0.20-0% (w/v), was used in the preparative scale and resulted in similar fractionation. The preparative-scale CPC has a superior sample loading capacity by the use of tubular dialysis membrane inside convoluted tubing as the separation channel. In this study, the quantity of the sample loaded into the preparative CPC was about 15 times more than that in the bench scale, and in a single run the preparative CPC could prepare approximately 3 mg of plasmid DNA with about 96% of RNA removed. The higher surface area per length of the separation channel in the preparative CPC was believed to benefit mass transfer of CTAB across the membrane, leading to less CTAB being required in the process.

Centrifugation↗
PubMed2006

Simultaneous reduction and alkylation of protein disulfides in a centrifugal ultrafiltration device prior to two-dimensional gel electrophoresis.

Reduction and alkylation of protein disulfides prior to IEF, when performed directly in a centrifugal ultrafiltration device, provides an effective means of terminating the alkylation reaction, concentrating the proteins for analysis, and removing ionic impurities that interfere with IEF. When cells were lysed in "buffers" that support the activity of enzymes such as lysozyme and benzonase, the conductivity of the resulting lysate was an order of magnitude higher than when lysis was induced by chaotropic urea detergent solutions. Following reduction and alkylation, the conductivity of both lysates was lowered by ultrafiltration to the 0.1-0.2 mS/cm range in preparation for IEF. The detergent 3-(4-heptyl)phenyl 3-hydroxypropyl dimethylammonio propanesulfonate (C7BzO), which favors the solubilization of proteins, but which interferes with SDS equilibration and second dimension PAGE, was effectively removed by ultrafiltration and exchanged with CHAPS without measurable loss of protein. Disparate protein patterns of Rhodopseudomonas palustris lysates were revealed by two-dimensional gel electrophoresis depending on which reagent was used to induce cell lysis.

Alkylation↗