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At least 883 records · Page 49Linked to original sources
PubMed1984

A centrifugal elutriation system of separating small numbers of cells.

One of the major disadvantages of centrifugal elutriation (CE) is the relatively large volume (150 ml) of the various fractions, especially if small numbers of cells have to be separated and the fractions contain few cells. To reduce the volume of the fractions 2 elutriator rotors were coupled in series. Since the rotor speed of the second rotor was always kept 750 rpm higher than that of the first rotor, cells elutriated from rotor 1 were collected in rotor 2. After elutriation of a complete fraction from rotor 1, and collection in rotor 2, the cells were harvested from rotor 2. This was achieved by means of a flow distribution unit (FDU), which made it possible to disconnect the flow of both rotors and simultaneously reverse the flow of the second rotor. It is demonstrated that 40-95 X 10(6) mononuclear leukocytes may be fractionated without loss of resolution in fractions of only 9 ml. The lymphocyte (greater than 99%) and monocyte subpopulations (88-94%) obtained were as pure as with CE carried out with only 1 rotor. In addition, the cells in rotor 2 could be washed and suspended in culture medium prior to harvesting by means of the FDU. In this way loss of cells by additional centrifugation steps was avoided. Erythrocytes (RBC) present in certain lymphocyte fractions were lysed with NH4Cl and after lysis of the RBC and elution of ghosts and debris, the cells were washed and harvested. This procedure did not affect cell viability and the PHA response of the lymphocytes. The versatile system described made it possible to apply CE for the separation of small numbers of cells without loss of resolution, and demonstrated that CE is ideally suitable for concentration and washing of cells, and removal of contaminating RBC, not affecting the recovery, viability and function of the cells.

Air Pressure↗
PubMed1984

Centrifugation of normal and rheumatoid arthritis blood on Ficoll-Hypaque and Ficoll-Nycodenz solutions.

Attempts to use the rapid single-step Ficoll-Hypaque centrifugation procedure for the purification of mononuclear and polymorphonuclear leucocytes from the blood of normal individuals and rheumatoid arthritis patients have sometimes been unsuccessful, largely because the erythrocytes would not sediment through the centrifugation medium. Re-evaluation of the factors (e.g. Ficoll concentration, temperature, and ratio of the diatrizoate salts) which affect these separations showed that under our conditions it was advantageous to use a medium with a lower viscosity (Ficoll concentration) and/or a higher osmotic strength (increased sodium diatrizoate: meglumine diatrizoate) than had been recommended previously (Ferrante and Thong, 1978; 1980; Ferrante et al., 1982). Higher osmotic strength media must be used for separating the components of blood from rheumatoid arthritis patients than from normal individuals because rheumatoid arthritis erythrocytes have a lower buoyant density than normal erythrocytes.

Arthritis, Rheumatoid↗
PubMed1986

A sensitive kinetic latex agglutination immunoassay adapted to centrifugal analysis.

Latex reagents for HCG obtained from commercially available pregnancy test kits were adapted for use on Instrumentation Laboratory's Multistat III Plus centrifugal analyzer. The clearance rate of an agglutinating reaction mixture can be measured by absorbance over a time period of 15 min. The automated system has a sensitivity of less than 5 mIU HCG/ml in buffer or urine with a range of up to 100 mIU HCG/ml. Statistical analyses on urine samples indicated CVs of between 7.6% and 10.7% for within-run precision and between 6.8% and 10.8% for between-run precision. The correlation between the centrifugal latex agglutination method and two commercially available RIAs was found to be about 87%.

Antibodies↗
PubMed1992

A rapid isolation procedure for dendritic cells from mouse spleen by centrifugal elutriation.

The standard isolation procedure for antigen presenting dendritic cells (DC) takes 2 days and includes selective adherence to tissue culture plates which may lead to the activation of these cells. This report describes the isolation of DC by centrifugal elutriation (CE). Murine spleen cells were separated on the basis of size and density into 7 CE fractions. This method took 90 min. Cells from each CE fraction were characterized by fluorescence activated cell sorter (FACS) analysis and their antigen presenting cell (APC) activity was determined by a secondary Sendai virus specific T cell proliferation assay. CE fraction 5 contained most of the DC with a concentration of 6-10%, representing an approximately 15-fold enrichment compared to unseparated spleen cells (< 1% DC). This CE fraction also exhibited the highest APC activity, which was almost completely abolished after depletion of DC by treatment with monoclonal antibody 33D1 (DC-marker) and complement. Further enrichment of CE fraction 5 by discontinuous density gradient centrifugation resulted in a cell population containing 35-55% 33D1-positive cells with similar characteristics as DC isolated by the standard procedure, such as the capacity to induce a primary viral peptide specific CTL response. Two-color FACS analysis showed an increase in MHC expression on 33D1-positive cells of CE fraction 5 after 18 h culture involving cell adhesion to a similar level as the MHC expression on DC isolated by the standard procedure. During this same period their morphology changed from a round to a dendritic appearance. In conclusion, our results indicate that CE is well suited for isolating DC more rapidly and without activation of these cells by adherence, a process which readily occurs in the standard isolation procedure.

Animals↗
PubMed1975

A technique for isolating and concentrating microfilariae from peripheral blood by gradient centrifugation.

A method for isolating microfilariae from blood cells by Ficoll-Hypaque centrifugation, followed by concentration by conventional centrifugation, is described. A 30 fold concentration and quantitative recovery of parasites was achieved. This technique should be of value for documenting low levels of parasitaemia, and for obtaining relatively pure suspensions of microfilariae for metabolic studies and antigen analysis.

Blood↗
PubMed1984

The superiority of the miniature Anion-Exchange Centrifugation Technique for detecting low grade trypanosome parasitaemias.

The efficiency was tested of the Haematocrit Centrifugation Technique (HCT) and the miniature Anion-Exchange Centrifugation Technique (mAECT) for demonstrating trypanosomes in the blood of four antelopes experimentally infected with Trypanosoma brucei gambiense in Liberia. During simultaneous daily application of both methods over a period of six months, parasitaemias were detected on 12 occasions by HCT and on 73 occasions by mAECT during 473 examination days, thus indicating the superiority of the mAECT for the detection of low grade trypanosome infections.

Animals↗
PubMed1989

Leucine aminopeptidase activity of polyploid cells after centrifugal elutriation of rat hepatocytes of various ages.

Rat hepatocytes were isolated by the perfusion method, and further separated with centrifugal elutriation into diploid cells and polyploid cells. The ploidy class and amount of polyploid (octaploid) cells increased with increasing flow rate. At higher flow rates of centrifugal elutriation, older rats contained much more octaploid cells than younger rats. Activity of acid phosphatase and leucine aminopeptidase increased with increasing flow rate (increasing ploidy class). Protein components of hepatocytes in octaploid cells were different from those of tetraploid cells. From these results, it is suggested that the function of the higher ploidy class hepatocytes is greater than that of lower ploidy class hepatocytes.

Acid Phosphatase↗
PubMed1996

Development of space motion sickness in a ground-based human centrifuge.

Adaptation of the vestibular system, specifically the otolith organs, to a non-terrestrial environment can result in space motion sickness-like symptoms when the human is reintroduced to the normal, 1 Gz, terrestrial environment. This premise was investigated by exposing nine subjects to 90 min of sustained 2 Gz acceleration in a human centrifuge and then observing and evaluating them at 1 Gz. Five of the subjects developed slight SMS symptoms, three developed moderate, and one developed frank sickness. Postural instabilities in two of the most affected subjects were also observed using the Equitest System post exposure. Long duration exposure to a non-terrestrial G(2Gz) appears to be a potential means for developing SMS-like symptoms in a ground-based human centrifuge.

Adaptation, Physiological↗
PubMed1984

The calcium uptake in smooth muscle microsomal vesicles is reduced by centrifugation.

A membrane fraction was isolated from the smooth muscle of the pig stomach by density gradient centrifugation. It was observed that the ATP-dependent Ca uptake in this fraction was diminished if the microsomes were pelleted by differential centrifugation. The decrease of the oxalate-independent Ca uptake was relatively small, but the oxalate-stimulated Ca uptake was reduced dramatically. Evidence is presented which indicates that the selective decrease of the oxalate-stimulated Ca uptake is mainly caused by mechanical damage of the vesicles. Since the oxalate-stimulated Ca uptake can be largely preserved by avoiding pelleting during the membrane fractionation, this observation may be very useful for the further study of Ca transport in subcellular fractions of smooth muscle.

Animals↗
PubMed1989

Isolation of cardiac ventricular myocytes from newborn rats by use of fractional elutriation centrifugation.

Isolation of cardiac ventricular myocytes from newborn rats (0-4 days old) by use of elutriation centrifugation is described. By the use of fractional centrifugation, a homogeneous myocyte population with high purity was obtained without any further procedures, yielding about 10(6) myocytes/rat. When 48-hr monolayer cultures were established, the cells showed normal pulsatory contractions. A morphological evaluation of such cultures is given.

Animals↗
PubMed1980

An airfuge centrifugation procedure for the measurement of ligand binding to membrane-associated and detergent-solubilized plasma membrane receptors.

A method is described in which high-speed centrifugation of membranes through an oil phase is used to separate membrane-bound and detergent-solubilized polypeptide receptor-iodinated ligand complexes from unbound ligands. Three centrifuges, the Brinkmann Eppendorf (5412), the Beckman Microfuge B and the Beckman Airfuge were evaluated for this capability. Under the conditions described, the Beckman Airfuge surpassed the others in recovering previously 125I- and 32P-labelled cell membranes. The Airfuge method was compared with the more classically employed membrane filtration method to measure specific [125I]insulin and [125I]thrombin binding to human placental membranes and an enriched plasma membrane fraction from mouse embryo fibroblasts, respectively, are found to be 4 to 6 times more sensitive. For example, specific binding of ligand to its receptor was demonstrated with 5 micrograms of protein. With slight modifications, the polyethyleneglycol 6000 method of precipitating 125I-labelled ligand-soluble receptor complexes can be adapted to the Airfuge sedimentation through oil procedure.

Animals↗
PubMed1984

An apparatus for counter-current distribution in a centrifugal acceleration field.

An apparatus is described that reduces the time required for counter-current distribution in aqueous polymer two-phase systems by a factor of more than five. Phase settling, which has been the time consuming step, is here facilitated by centrifugation. The crucial point in the construction, that allows automatization, is that the transfer step in the counter-current distribution cycle is performed during centrifugation.

Centrifugation↗
PubMed1988

The removal of exogenous thiols from proteins by centrifugal column chromatography.

Centrifugal column chromatography was shown to provide a rapid, efficient, and useful means of separation of various low molecular weight thiols from proteins. The single chromatographic step procedure employed standard 5 ml plastic syringes containing Sephadex G-25 as the bed matrix and required less than 5 min to produce average dilutions of 5000-, 980-, and 25-fold, respectively, from 5 to 200 mM initial concentrations of 2-mercaptoethanol, dithiothreitol, and reduced glutathione in the sample as measured by titration with 5,5'-dithiobis-(2-nitrobenzoic acid). Dihydrofolate reductase solutions of 0.07-0.08 mM were separated from 50 mM 2-mercaptoethanol, dithiothreitol, or reduced glutathione with a minimum 16,500-fold dilution of the thiol after centrifugal chromatography on two consecutive columns. Thymidylate synthase solutions of 0.06 mM were effectively separated from 50 mM 2-mercaptoethanol or dithiothreitol with a minimum average 5900-fold dilution of the thiol after consecutive column chromatography. There was no change in either the physical or chemical properties of the enzyme throughout the course of the experiments as determined by activity, active site sulfhydryl group titration, and binding assays. Recoveries of protein obtained in the load fraction were usually in excess of 70% of the protein loaded with virtually no dilution from the initial concentration. This method was developed in order to facilitate the study of the active site sulfhydryl groups in enzymes.

Centrifugation↗
PubMed1983

Correlation between MAO activity in blood platelets obtained by single and multiple centrifugations.

Monoamine oxidase (MAO) activity in human blood platelets isolated by a single centrifugation at 600g for 2 1/2 minutes, which isolates only 45-65% of total platelets, was highly correlated with the MAO activity in platelets isolated by a method which isolates essentially 100% of platelets (r = 0.95, p less than 0.01). These results indicate that the single centrifugation method, which isolates only a portion of platelets, gives a reliable estimate of MAO activity in the entire platelet population.

Blood Platelets↗
PubMed1983

Isolation of bovine polymorphonuclear leukocytes by density gradient centrifugation.

A method for the isolation of an enriched population (greater than 95%) of bovine polymorphonuclear leukocytes (PMNs) was developed using density gradient centrifugation. Leukocytes were isolated from peripheral blood by centrifugation in a density gradient medium (Percoll) of specific gravity 1.092. Viability was greater than or equal to 95% and the isolated PMNs were functional in migration inhibition and chemiluminescence assays. This has proved to be a simple effective method for obtaining bovine PMNs and yields cell populations that can be utilized for a variety of measures of PMN function.

Animals↗
PubMed1987

Preparation of nasopharyngeal secretions for immunofluorescence by one-step centrifugation through Percoll.

A simple method was developed for separation of cells from nasopharyngeal secretion for the diagnosis of respiratory virus infections by immunofluorescence microscopy. The diluted specimen, containing dithiothreitol to break up the mucus, was centrifuged once through a cushion of 20% Percoll (colloidal silica, a density gradient medium), which permitted sedimentation of cells through the cushion, but retained mucus on top of it. The pelleted cells were resuspended, and microscope slides were then prepared by standard techniques. The Percoll centrifugation method was also applicable for sputum and bronchoaveolar lavation specimens. Immunofluorescent antibody staining of nasopharyngeal secretions prepared by the described method was more sensitive than enzyme immunoassay for the detection of respiratory syncytial virus.

Antigens, Viral↗
PubMedDate not supplied

Centrifugal enhancement of human immunodeficiency virus (HIV) and human herpesvirus type 6 (HHV-6) infection in vitro.

The effect of centrifugal inoculation of human immunodeficiency virus (HIV) and human herpesvirus-6 (HHV-6) on the infectivity of the viruses for cell cultures was examined. Three HIV-1 strains, ARV-2, HTLV-IIIb and a local isolate, WA-46c, were tested in peripheral blood lymphocytes, HUT-78, H9 and MT-2 cells. The HHV-6 strain was a local isolate and was studied only in peripheral blood lymphocyte cultures. Centrifugal inoculation of the viruses at a force of 2500 x g for 60 min, enhanced HIV-1 infectivity by a factor of about 10-fold in all cell cultures tested. Infectivity was increased about 100-fold for HHV-6.

Cell Line↗
PubMed1989

Increased sensitivity for rapid detection of cytomegalovirus by shell vial centrifugation assay using mink lung cell cultures.

A comparative study was made of various human and non-human cell cultures to determine their sensitivity for cytomegalovirus (CMV) as detected by the production of CMV early antigen using the shell vial centrifugation assay. Mink lung cell cultures, frequently used for detection of herpes simplex virus in clinical specimens, were found to be significantly more sensitive to infection by CMV than other cell cultures tested. Using the shell vial centrifugation assay, the mink lung cell cultures were more sensitive than human diploid fibroblasts for the detection of the Davis strain of human CMV and CMV from clinical specimens.

Animals↗