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At least 937 records · Page 52Linked to original sources
PubMed1997

Separation by counterflow centrifugal elutriation and analysis of T- and B-lymphocytic cell lines in progressive stages of cell division cycle.

The method of counterflow centrifugal elutriation (CCE) facilitates the non-invasive separation of proliferating cells into the progressive stages of the cell division cycle. We present here detailed protocols for the separation of primary lymphocytes and lymphocytic cell lines including Jurkat, a mature human T-cell line, Ramos, a human B-cell line, WEHI-231, a murine B-cell lymphoma, and stimulated human peripheral T-cells into progressive stages of the cell division cycle by counterflow centrifugal elutriation. Protocols for using the elutriator to concentrate large volumes of cells prior to separation, the preparation of highly enriched lymphocyte populations at progressive stages through the cell division cycle and conversion parameters from low to high volume rotors are described. Simple dual-staining methods of BrdUrd incorporation and propidium iodide staining for DNA content and subsequent flow cytometry are detailed. Together with [3H]thymidine incorporation data these provide a very accurate determination of cell cycle position of the separated populations.

Animals↗
PubMed1984

The cell in the field of gravity and the centrifugal field.

It appears that the literature and logic that the earth's gravity has been one factor in the limitation of cell size, as well as being an important influence on the diversity of cell types and sizes throughout biological evolution. Analysis of the literature reveals an inverse relationship between the centrifugal force needed for intracellular stratification and cell size. The cells studied ranged in size from approximately 1 mm (amphibian eggs, Pelomyxa) to 0.01 mm (erythrocyte, lymphocyte), and g-forces ranged from about 100 g to 100 000 g respectively. Stratification within cell nuclei and organelles requires even greater forces, presumably because of their smaller size. Extrapolation from centrifugal forces to the force of gravity, and from the full stratification to the initial sedimentation of cell parts suggests a hypothesis for the evolutionary survival and existence of cells in the field of gravity. Average cell size results, in part, from the physical equilibrium between the destructive influence of the force of gravity and the protective role of diffusion and the cytoskeleton. At increased forces of gravity the cell size would thus be decreased, whereas at lower gravitational forces and weightlessness cell size would be expected to increase. Mechanisms of protection of giant cells against internal sedimentation are based on protoplasmic motion, thin and elongated shape of the cell body, increased cytoplasmic viscosity, and a reduced range of specific gravity of cell components, relative to the ground-plasm. The nucleolus, due to its higher density, is considered as a possible trigger of mitosis.

Animals↗
PubMed1977

The measurement of androgen receptors in human prostatic tissue utilizing sucrose density centrifugation and a protamine precipitation assay.

Methods have been established for the measurement of androgen receptors in the cytosol and crude nuclear extract of human prostatic tissue using sucrose density gradient centrifugation and a protamine precipitation assay. These studies have demonstrated the presence of a high affinity, heat labile protein that binds preferentially to dihydrotestosterone and that is present only in androgen target tissues. In studies of prostatic tissue from 15 patients sucrose density gradient centrifugation appeared more sensitive than the protamine precipitation assay in the identification of androgen receptors. These findings appear promising and suggest that in the near future a reliable assay may be available for the measurement of androgen receptors in prostatic carcinoma.

Centrifugation, Density Gradient↗
PubMed2001

Errors in bound water measurements using centrifugal settling method.

Centrifugal settling method (CSM) is commonly used to estimate the bound water content in sludges. The sludge sediment matrix should be purely plastic for accurate measurements to the bound water content. This communication, however, addressed for the first time that, owing to the inhibited elasticity of the sludge matrix, sediment height rebound was commonly observed for the flocculated sludge. Also, different sludges could have distinct elastic responses after centrifugation. There existed no simple correlation to incorporate the elastic rebound of sludge sediment in bound water measurement. The possible errors incorporated into CSM for measuring bound water contents were highlighted.

Centrifugation↗
PubMed2002

Comment on "experimental analysis of centrifugal dewatering process of polyelectrolyte flocculated waste activated sludge".

In summary, after supernatant liquid was exhausted in the author's experiments, menisci formed, and shrinkage, not pore evacuation and drying, was the mechanism causing removal of water thereafter. Results of centrifugal filtration experiments cannot be applied to solid bowl centrifugation because solid compression conditions are quite different in the two types of machines.

Centrifugation↗
PubMedDate not supplied

Artificial gravity: head movements during short-radius centrifugation.

Short-radius centrifugation is a potential countermeasure to long-term weightlessness. Unfortunately, head movements in a rotating environment induce serious discomfort, non-compensatory vestibulo-ocular reflexes, and subjective illusions of body tilt. In two experiments we investigated the effects of pitch and yaw head movements in participants placed supine on a rotating bed with their head at the center of rotation, feet at the rim. The vast majority of participants experienced motion sickness, inappropriate vertical nystagmus and illusory tilt and roll as predicted by a semicircular canal model. However, a small but significant number of the 28 participants experienced tilt in the predicted plane but in the opposite direction. Heart rate was elevated following one-second duration head turns. Significant adaptation occurred following a series of head turns in the light. Vertical nystagmus, motion sickness and illusory tilt all decreased with adaptation. Consequences for artificial gravity produced by short-radius centrifuges as a countermeasure are discussed. Grant numbers: NCC 9-58.

Adaptation, Physiological↗
PubMed1989

Rapid diagnosis of malaria by acridine orange staining of centrifuged parasites.

A rapid diagnostic test for malaria based on acridine orange staining of centrifuged parasites in a microhaematocrit tube ('QBC' tube) was compared with the thick blood smear in 12 volunteers experimentally infected with Plasmodium falciparum, 408 residents of a malaria endemic area, and 180 hospital patients with suspected malaria. In the experimentally infected volunteers, the QBC tube test and the thick blood smear were comparable and the QBC tube could detect as few as 4 parasites/microliter blood. When used for mass screening in the field study, the test had a sensitivity of 70% for the diagnosis of malaria compared with 92% for a single thick blood smear. However, when used to diagnose malaria in hospital patients, the test detected as few as 3 parasites/microliter in 91 of 92 patients with asexual parasitaemia. For the three studies, the QBC tube was highly specific (98.4%), indicating malaria in only 8 of 487 subjects with negative blood films. The species of parasite was correctly identified in 77% of species. Processing the QBC tube was easier and much more rapid than was processing a thick blood smear, taking only 5 min for centrifugation and 5 min for examination. The QBC tube is not a substitute for the blood smear, but its speed and ease of use make it an important new tool for the diagnosis of malaria.

Acridine Orange↗
PubMed1999

Susceptibility of human and non-human cell lines to HCV infection as determined by the centrifugation-facilitated method.

The centrifugation-facilitated inoculation method was used to test 51 human and non-human cell lines for ability to support HCV replication. As determined by nested RT-PCR, one fifth of the cell lines tested were virus positive 15 days post inoculation suggesting that the centrifugation-facilitated inoculation is an efficient method for cell infection with HCV. However, virus production by infected cultures remained of low grade, thus showing that the unknown factors which limit HCV replication in vitro are not overcome by the procedure.

Amino Acid Sequence↗
PubMed2003

Evaluation of two recovery methods for detection of Mycobacterium avium subsp. paratuberculosis by PCR: direct-dilution--centrifugation and C(18)-carboxypropylbetaine processing.

A duplex polymerase chain reaction (PCR)-hybridization assay based on Mycobacterium avium subsp. paratuberculosis (MAP)-specific IS900 integration sites was used to evaluate two mycobacterial recovery methods from bovine feces: a direct-dilution-centrifugation method and a C(18)-carboxypropylbetaine (CB-18)-based method. All MAP PCR results were confirmed for absence of inhibitors using a novel PCR system based on the rpoB gene of plant chloroplasts as an internal control. The detection limits of both MAP recovery methods when coupled with PCR were determined to be between 100 and 1000 organisms. Using culture as a 'gold standard' PCR following the direct-dilution-centrifugation protocol was 92.6% sensitive and 83.7% specific, whereas PCR following the CB-18 method was 100% sensitive and 53.5% specific. Both methods were 100% specific when 60 'true' negatives from two uninfected herds were tested. Both the CB-18 and direct processing methods coupled with a target-specific amplification technique may provide greater sensitivity to diagnose subclinical animals as they were able to detect more positives, on samples derived from infected herds, than conventional culture methods; however, more extensive investigation and follow-up of suspect animals will be required to fully validate the MAP recovery and molecular detection protocols described.

Animals↗
PubMed2002

Detection of Mycobacterium avium subsp. paratuberculosis by buoyant density centrifugation, sequence capture PCR and dot blot hybridisation.

Detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) by polymerase chain reaction (PCR) is often hampered by the lack of efficient methods for sample treatment. We report a protocol for analysis of faecal samples based on buoyant density centrifugation in Percoll and IS900 sequence capture PCR combined with a dot blot assay for detection of low-grade infection of M. paratuberculosis. Serial dilutions of M. paratuberculosis genomic DNA and M. paratuberculosis bacteria were used to assess the sensitivity of the method. The final evaluation was performed with spiked faecal samples, which also were analysed by culture. The presence of PCR inhibitory substances in processed faecal samples was evaluated by including a PCR internal control. By using buoyant density centrifugation, sequence capture PCR, and dot blot hybridisation, we achieved a sensitivity of 10(3)CFU (colony forming units)/g of faeces. The detection limit by culture was assessed to 10(2)CFU/g of faeces. We conclude that the described protocol is a fast and sensitive alternative to bacterial culture of faecal samples.

Animals↗
PubMed1984

Continuous extraction of urinary anthracycline antitumor antibiotics with the horizontal flow-through coil planet centrifuge.

Extraction of doxorubicin (adriamycin) and daunorubicin and their metabolites from human urine was attempted utilizing the horizontal flow-through coil planet centrifuge. Partition coefficients of the drugs for various combinations of non-aqueous phases and aqueous salt solutions were determined. Optimal coefficients for adriamycin and daunorubicin were achieved with n-butanol-0.3 M disodium hydrogen phosphate. Extraction efficiencies of the drugs from human urine comparable to those obtained by standard resin column techniques could be realized by employing the n-butanol-urine (containing 0.3 M disodium hydrogen phosphate) system in the coil planet centrifuge, at flow-rates of 500-600 ml/h, and at 650 rpm revolutional speed. Small quantities of drugs and metabolites could be continuously concentrated into small volumes of the n-butanol phase from large volumes of salted urine. The versatility of the technique was demonstrated by its application to extraction of aclacinomycin A, a novel anthracycline antitumor agent, and its metabolites from human urine.

1-Butanol↗
PubMed1998

Centrifugal partition chromatographic reaction for the production of chiral amino acids.

The use of a centrifugal partition chromatographic reactor is investigated for the production of chiral amino acids from racemic mixtures. Chirally selective enzymatic hydrolysis of N-acetyl-L-methionine into acetic acid and L-methionine was carried out in the chromatographic reactor to demonstrate the concept of integrated reaction and separation in centrifugal partition chromatography (CPC). The products L-methionine and acetic acid, as well as the unconverted substrate, N-acetyl-D-methionine are obtained separately. An aqueous two-phase system, consisting of PEG 600, potassium phosphate and water was successfully applied as liquid-liquid two-phase system in CPC. A model is presented, which describes the reaction chromatograms on the basis of the independently measured partition and mass transfer coefficients of the individual (reacting) components. The model appears to be a valuable tool for optimizing the reaction-separation process.

Amino Acids↗
PubMed1998

Enhancement of varicella-zoster virus detection in A-549 shell vials by use of freeze-thawed specimens, extended incubation, and "a centrifuged, not incubated" direct detection method.

A total of 95 clinical samples were cultured for periods of 2 and 7 days in centrifuged A-549 shell vials before and after freezing and thawing of specimens. In addition, centrifuged A-549 shell vials were tested directly for varicella-zoster virus without incubation using a direct fluorescent antibody (DFA) technique. Twenty-seven specimens were positive by at least one method. The sensitivity for DFA on unincubated A-549 shell vials was 85.2%; for unfrozen 2-day cultures, 88.9%; for unfrozen 7-day cultures, 92.6%; for freeze-thaw 2-day cultures, 92.6%; and for freeze-thaw 7-day cultures, 96.3%. Freeze-thawed specimens cultured for 7 days yielded the highest number of positive results with conspicuous cell-to-cell spread as a sign of viral replication.

Cell Line↗
PubMed2001

Influence of the centrifugal granulating process on the properties of layered pellets.

Drug-layered pellets based on microcrystalline cellulose (MCC) beads as substrates were prepared using a laboratory-scale centrifugal granulator. The effect of three independent process parameters (rotor rotation speed, slit air flow rate, and spray air rate) on responses describing the amount of drug loss during the process, amount of agglomerates, bulk density, flowability, friability, shape, and surface roughness were studied using a 3(3) full factorial experimental design. The variables studied were found to have a significant influence on the responses evaluated. Rotor rotation speed and slit air flow rate had a significant positive influence on the amount of drug loss during the process and the amount of agglomerates, whereas rotor rotation speed and spray air rate had the same effect on the bulk density, flowability, and the roundness of the pellets. The amount of agglomerates and the roundness value of the pellets were negatively affected by the spray air rate while the slit air flow rate showed the same effect on the bulk density and flow rate of the pellets. In addition to the main effects, there were some significant paired interactions between slit air flow rate and spray air rate as well as rotor rotation speed and slit air flow rate. Based on the results, the significance of these three parameters should be considered carefully for quality pellet preparation by the centrifugal granulating technique using MCC beads as substrates.

Cellulose↗
PubMed2001

Plasma membrane NADH oxidase of maize roots responds to gravity and imposed centrifugal forces.

NADH oxidase activities measured with excised roots of dark-grown maize (Zea mays) seedlings and with isolated plasma membrane vesicles from roots of dark-grown maize oscillated with a regular period length of 24 min and were inhibited by the synthetic auxin 2,4-dichlorophenoxyacetic [correction of dichorophenoxyacetic] acid. The activities also responded to orientation with respect to gravity and to imposed centrifugal forces. Turning the roots upside down resulted in stimulation of the activity with a lag of about 10 min. Returning the sections to the normal upright position resulted in a return to initial rates. The activity was stimulated reversibly to a maximum of about 2-fold with isolated plasma membrane vesicles, when subjected to centrifugal forces of 25 to 250 x g for 1 to 4 min duration. These findings are the first report of a gravity-responsive enzymatic activity of plant roots inhibited by auxin and potentially related to the gravity-induced growth response.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1998

Tests on the centrifugal flotation technique and its use in estimating the prevalence of Toxocara in soil samples from urban and suburban areas of Malaysia.

The influence of soil texture (silt, sand and laterite) and flotation solutions (saturated NaCl, sucrose, NaNO3, and ZnSO4) upon the recovery of Toxocara ova from seeded soil samples with the centrifugal flotation technique was investigated. Soil samples of different texture were artificially seeded with Toxocara spp. ova and subjected to a centrifugal flotation technique which used various flotation solutions. The results showed significant (P < 0.001) interactions between the soil types and the flotation solutions. The highest percentage of ova recovery was obtained with silty soil (34.9-100.8%) with saturated NaCl as the flotation solution (45.3-100.8%). A combination of washing of soil samples with 0.1% Tween 80, and flotation using saturated NaCl and a 30 min coverslip recovery period was used to study the prevalence of contamination of soil samples. Forty-six soil samples were collected from up to 24 public parks/playgrounds in urban areas of Petaling Jaya and suburban areas of Serdang. The prevalence of Toxocara species in the urban and suburban areas was 54.5% and 45.8% respectively.

Animals↗
PubMed1981

Separation of Isospora (Toxoplasma) gondii cysts and cystozoites from mouse brain tissue by continuous density-gradient centrifugation.

A simple, quick and reproducible method consisting of density-gradient centrifugation of homogenized infected mouse brain tissue on Percoll is described for the isolation and purification of cysts of Isospora (Toxoplasma) gondii. A 100% recovery of cysts, with 74.2% in a single fraction with a specific gravity of 1.056, was obtained by overlaying homogenates of infected mouse brains on a pre-formed Percoll gradient and centrifugation at low g forces. With this procedure recovery was independent of the age of the cysts. Titration of purified cystozoites showed there to be no loss of infectivity.

Animals↗
PubMed1993

Simple centrifugation method for efficient pelleting of both small and large unilamellar vesicles that allows convenient measurement of protein binding.

Separation of unilamellar model membrane vesicles from external solution is often an important step in quantitation of vesicle bound or entrapped materials. An efficient method that allows pelleting of both small and large model membrane vesicles by centrifugation is described in this report. In this method streptavidin is added to vesicles containing a trace amount of biotinylated lipid. The resulting aggregation allows pelleting of the vesicles using an ordinary high-speed centrifuge. Control experiments show that the addition of streptavidin does not induce substantial vesicle fusion or leakage of substances trapped in the internal aqueous compartment of the vesicles. The method can accommodate different phospholipid compositions and lipid concentrations. Experiments with proteins that switch between hydrophilic and hydrophobic states show that the method can readily be used to monitor protein binding to vesicles.

Adenosine Triphosphatases↗