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PubMed1983

Separation of different cell populations of rat liver by density gradient centrifugation in a vertical rotor with self-generated Percoll gradients.

A procedure is described which, with good resolution, separates viable rat liver parenchymal cells from other cell populations present in crude suspensions of liver cells. Fractionation is carried out by vertical rotor centrifugation in a self-generated Percoll gradient. Kupffer cells can be separated from other sinusoidal cells, resulting in a separate peak of peroxidase negative non-parenchymal cells, composed mainly of endothelial cells.

Animals↗
PubMed1965

NEW METHOD FOR PURIFYING COXIELLA BURNETII BY DEXTRAN SULFATE PRECIPITATION FOLLOWED BY CENTRIFUGATION.

This paper describes a simple method for purifying Coxiella burnetii cultivated in the chick embryo. The procedure is based upon the preliminary clarification of infected whole-egg suspensions by precipitating the low-density lipoproteins and other extraneous solids with dextran sulfate, calcium chloride, and sodium chloride solutions, and then concentrating the rickettsiae by centrifugation.

Animals↗
PubMed1965

PLASMA TRAPPING IN THE CENTRIFUGED RED CELLS OF CHILDREN WITH SEVERE THALASSAEMIA.

The amount of trapped plasma, expressed as a percentage of the packed cell volume, was determined in samples of heparinized blood from 26 children with severe thalassaemia and from 37 normal children over a wide range of artificial packed cell volumes. There was invariably more plasma trapped among the centrifuged red cells of the thalassaemic children, the mean difference between normal and thalassaemic children being 1% of the packed cell volume. In both normal and thalassaemic children the volume of trapped plasma, expressed as a percentage of the packed cell volume, was directly proportional to the packed cell volume. Plasma trapping did not appear to be related to the size of the erythrocytes in either the normal or the thalassaemic children. It varied inversely with the density of the red cells, as estimated by their mean haemoglobin concentration, in the thalassaemic children.

Adolescent↗
PubMed1986

Gel electrophoresis of proteoglycan monomers of baboon articular cartilage separated by zonal rate centrifugation in sucrose gradients.

Proteoglycan monomers from articular cartilage of young baboons and of a baboon foetus were submitted to zonal rate centrifugation in sucrose gradients. The peaks obtained were analyzed by gel electrophoresis on polyacrylamide-agarose gels. The proteoglycan monomers corresponding to the bands obtained by gel-electrophoresis of unfractionated proteoglycans were separated in the gradients and the sedimentation distance correlated with the electrophoretic mobility.

Animals↗
PubMed1986

Isolation by Percoll gradient centrifugation and radiolabeling of bloodstream forms of Trypanosoma cruzi.

A method to isolate T. cruzi bloodstream forms was designed taking advantage of Percoll self-generated gradients and isopycnic centrifugation, resulting in a good resolution between parasites and host cells. Purified parasites incorporated 3H-uridine giving values of 23% precipitable by TCA. Viability of the parasites was totally preserved as evidenced by the "in vivo" infectivity assays.

Animals↗
PubMed1978

A two-step centrifugation procedure for the purification of sheep erythrocyte antigen-binding cells.

A two-step centrifugation procedure has been developed to isolate greater quantities of highly purified sheep erythrocyte antigen-binding cells (ABC) than previously possible. The first step involves partially separating sheep erythrocyte rosettes from unrosetted lymphocytes by their difference in buoyant density on Ficoll-Hypaque. Subsequent passage through a linear 5 to 10% Ficoll gradient produces further purification of rosettes by sedimentation velocity. Approximately 4.5 X 10(6) ABC can be obtained at 50 to 100% purity from 10(9) immune spleen cells (5 days post-immunization) and 1 X 10(5) ABC at 20 to 40% purity from 10(9) nonimmune spleen cells. The purified ABC from 5-day immune animals are 80 to 90% B cells and 10 to 20% T cells, and represent between 30 and 40% of the original ABC in the spleen cell population. Less than 0.2% of the purified ABC are plaque-forming cells (PFC) and less than 2% have intracellular immunoglobulin (Ig) or J chain. The quantities of ABC obtained are sufficient for investigating biochemical parameters of antigen-induced lymphocyte activation and for direct analysis of the surface isotypes found on antigen-binding cells after immunization.

Animals↗
PubMed1980

Ultrastructure and physical characteristics of proplastids and chloroplasts isolated by zonal centrifugation from Euglena gracilis var. bacillaris.

Proplastids at various stages of development and mature chloroplasts were isolated by zonal centrifugation in a double-sigmoid, sucrose-sorbitol gradient from Euglena gracilis var. bacillaris grown in darkness and subjected to various periods of illumination with white light. Computer programs were used in characterizing physical properties of the isolated organelles and in predicting the location of plastids of different developmental stages in the gradient. The technique permits computation of these parameters in any gradient material. The convoluted nature of the early developing plastids, revealed by electron microscopy, suggests that the frictional coefficients should be taken into account in describing the physical constants of the organelles, since their sedimentation coefficients are lower than would be expected for spherical particles of the same size and density.

Animals↗
PubMed1996

Identification and characterization of various differentiative growth plate chondrocytes from porcine by countercurrent centrifugal elutriation.

Countercurrent centrifugal elutriation was used to separate growth plate chondrocytes from porcine basing on their differences in sizes and densities. Eighteen fractions of cells with different sizes and densities were obtained. The mean cellular volumes increased progressively in each of successive fractions, and that increase was associated with specific phenotypic changes, such as biochemical differences in DNA synthesis, proteoglycan synthesis, and activities of alkaline phosphatase. Three distinct chondrocyte subpopulations with their unique characteristics were identified among the elutriated fractions. The resting chondrocytes were found to be small in size and quiescent. The hypertrophic chondrocytes were found to be large in size and metabolically active both in alkaline phosphatase and in proteoglycan productions. The proliferative chondrocytes exhibited a high DNA synthesis rate, and their sizes were found to be between those of the resting and hypertrophic chondrocytes.

Alkaline Phosphatase↗
PubMed2001

Proteomic analysis of human plasma: failure of centrifugal ultrafiltration to remove albumin and other high molecular weight proteins.

Determination of specific low abundance proteins, usually by radiolabelled or enzyme-linked immunoassays in serum or plasma is widely used in diagnostic medicine. Substitution of these assays by a proteomic approach has been suggested, but this methodology has far from realised its potential as a diagnostic tool. The main protein fractions of plasma represent more than 80% of total protein, making the hundreds or even thousands of other proteins difficult to detect by two-dimensional electrophoresis (2-DE). Thus, loading sufficient sample to detect trace proteins invariably means excessive loading of albumin and other high abundance proteins. The aim of this study was to determine whether centrifugal ultrafiltration of whole plasma could be used to eliminate proteins exceeding a desired molecular weight cut-off. Cellulose filters with a 30 kDa molecular weight cut-off were used with whole plasma, and total protein was determined before and after ultrafiltration. Samples were processed by routine methods for 2-DE using 18 cm, pH 3-10 isoelectric focusing strips for the first dimension and 7-15% gradient gels for the second dimension followed by silver staining. Gel analysis of the retentate fraction (> 30 kDa) revealed a typical 2-DE plasma profile with most of the major landmark proteins in place and as expected, the gels lacked many of the smaller (< 30 kDa) proteins. Comparison with gels of the filtrate fraction (> 30 kDa) revealed very little difference. Not only were many of the higher molecular weight proteins still present, but some of the smaller < 30 kDa landmark proteins were absent. Overall, gels of both the retentate and filtrate fractions were less informative than gels of whole plasma.

Blood↗
PubMed1979

The plasma kinetics of hydroxyethyl starch 350/0.60: a potential new adjunct for centrifugal leucapheresis.

A modified species of hydroxyethyl starch (HES 350/0.60) possessing a Mw- of 350,000 daltons combined with a molar hydroxyethyl group substitution (MS) of 0.60 (60 hydroxyethyl groups.100 glucose residues) was clinically assessed in seven normal subjects to determine the influence of these chemical modifications on intravascular clearance kinetics concomitantly with effects on the suspension stability (ESR) of blood. Following a standardised intravenous dose (30 gm.m2 BSA), the concentration of HES 350/0.60 in serum fell to half its peak value in 11.8 +/- 1.3 (SD) hours, while the ESR remained elevated for up to 12 hours post-injection. By adopting a Mw- of 350,000 daltons, the critical molecular weight (Cmw) of this colloid was surpassed, while the critical concentration (Cc), below which the suspension stability of blood is not affected, was shown to range between 0.3 and 0.5 gm.dl-1. In comparison to the present species of HES (Mw- 450,000 daltons, MS: 0.70) utilised as a sedimenting agent duirng centrifugal leucapheresis, HES 350,000/0.60 appears to affect the ESR in a similar manner, but is removed from the intravascular space approximately twice as rapidly. This more rapid clearance should be useful in avoiding cumulative build-up of HES in blood concomitant with reducing the total amount of intravascular H2O bound to this colloid, in normal and CML donors undergoing multiple cell collection procedures.

Adult↗
PubMed1986

Depletion of T lymphocytes from human bone marrow by the use of counterflow elutriation centrifugation.

Normal donor bone marrow buffy coat (BMBC) cells were fractionated into three subsets by counterflow elutriation centrifugation. Fraction 1 (Fr-1) was lymphocyte-enriched, fraction 2 (Fr-2) was a mixture of lymphocytes and myelomonocytic cells, and fraction 3 (Fr-3) was enriched for myelomonocytic cells. The extent of T cell depletion was determined by limiting dilution analysis of the growth of T cells. The cells in each group were stimulated with phytohemagglutinin and cultured in the presence of interleukin-2. These conditions allowed clonogenic growth of one of three T cells purified from peripheral blood. After a 3-week culture period, replicate wells were scored for growth and precursor frequency was determined. The frequency of T cells in BMBC was 1/22 (n = 4), 1/16 in FR-1 (n = 4), and 1/246 in Fr-2 (n = 2), and in Fr-3 it ranged from 1/3,000 to 1/10,000 (n = 4). For comparison, depletion of T cells from bone marrow by cytofluorographic sorting with OKT-11 and twice E-rosetting yielded 98.1% and 97.4% removal of T cells, respectively. Thus, the clonogenic assay assured that more than 99.3% of the original T cells were depleted by elutriation; however, hematopoietic progenitor assay (CFU-GM, CFU-GEMM, and BFU-E) showed no enrichment in any particular fraction, implying that the progenitors measured in this assay are of diverse sizes. Elutriation represents a useful method for depletion of T cells from human bone marrow. Limiting dilution assay of T cell growth is a sensitive measure to monitor T cell depletion.

Bone Marrow Cells↗
PubMed2002

Albumin recovery with centrifugal adsorption technology (CAT).

Centrifugal adsorption technology (CAT) is a new compact, countercurrent technology for efficient adsorption from large liquid streams by using adsorbent particles in the micrometer range. CAT seems particularly suited for the recovery of macromolecules at low concentrations, because the small particle dimensions lead to fast mass transfer rates. In this work, the potential of CAT for protein recovery is studied by model and experiment. A predictive model for the separation performance of CAT is presented, incorporating mass transfer resistance and axial dispersion transport in the liquid and the adsorbent phases. The model calculations were compared to experimental data for the adsorption of bovine serum albumin (BSA) on a standard commercial anion-exchange resin with particle diameter d(p) = 50 microm in a pilot-scale CAT apparatus. The model calculations accurately predicted the separation efficiency of CAT. The experimental set-up is shown to be mass transfer limited for the conducted experiments, which agrees with the model predictions. The model was also used to estimate the dimensions and performance of a CAT apparatus for the large-scale recovery of human serum albumin (HSA) from fermentation broth at the scale of 40 tons per year. The resulting equipment dimensions proved to be very small indeed, making CAT a potentially very attractive technology.

Adsorption↗
PubMed2004

Fractionation of protein, RNA, and plasmid DNA in centrifugal precipitation chromatography using cationic surfactant CTAB containing inorganic salts NaCl and NH(4)Cl.

Centrifugal precipitation chromatography (CPC) is a separation system that mainly employs a moving concentration gradient of precipitating agent along a channel and solutes of interest undergo repetitive precipitation-dissolution, fractionate at different locations, and elute out from the channel according to their solubility in the precipitating agent solution. We report here for the first time the use of a CPC system for fractionation of protein, RNA, and plasmid DNA in clarified lysate produced from bacterial culture. The cationic surfactant cetyltrimethylammonium bromide (CTAB) was initially used as a precipitating agent; however, all biomolecules showed no differential solubility in the moving concentration gradient of this surfactant and, as a result, no separation of protein, RNA, and plasmid DNA occurred. To overcome this problem, inorganic salts such as NaCl and NH(4)Cl were introduced into solution of CTAB. The protein and RNA were found to have higher solubility with the addition of these salts and separated from the plasmid DNA. Decreasing surface charge density of CTAB upon addition of NaCl and NH(4)Cl was believed to lead to lower surfactant complexation, and therefore caused differential solubility and fractionation of these biomolecules. Addition of CaCl(2) did not improve solubility and separation of RNA from plasmid DNA.

Centrifugation↗
PubMed1979

High-resolution flow-zonal centrifuge system.

A modified CF-32 Beckman flow centrifuge rotor has been developed that provides a long sedimentation path length with high gravitational force at the gradient sample interface. The modified rotor exhibits excellent separative capability and extraction efficiency when applied to purification of human influenza B and herpes simplex viruses.

Centrifugation, Zonal↗
PubMed1986

Separation of the components of commercial digitonin using high-performance liquid chromatography and centrifugal countercurrent chromatography. Identification of the products by californium-252 plasma desorption mass spectrometry.

Commercial digitonin has been separated into its components using high-performance liquid chromatography and centrifugal countercurrent chromatography. The individual glucosides have been identified by californium-252 plasma desorption mass spectrometry, occasionally supplemented by hydrolysis and analysis of the liberated sugars and aglycones. Mass spectra of commercial digitonin that have appeared in the literature are discussed in the light of their complex nature. A new glycoside of molecular weight 1035 is described and a structure proposed.

Californium↗