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Density gradient centrifugation using Ficoll-Conray solution was attempted to isolate viable canine transmissible sarcoma (CTS) cells. The viability of isolated tumor cells increased from about 50% to > 90%, and the yield of CTS cells was > 50% with over 99% purity, in when an isolation solution density of 1.05 was used.
The density profile of mouse bone marrow megakaryocytes, as determined by discontinuous albumin density gradient centrifugation, was characterized by a single density population (1.088 to 1.119 g/ml) with a peak density of 1.10 g/ml and a maximum enrichment of 15.5. This single population comprised both mature and immature megakaryocytes. The density profile of small, acetylcholinesterase-positive cells, a class of potential megakaryocyte precursors, was almost identical to that of morphologically recognizable megakaryocytes.
Alveolar type II cells were isolated from five human lung specimens obtained during resection or lobectomy and enriched to 63-85% purity. Digestion with Sigma protease type XIV followed by centrifugal elutriation and Percoll density gradient centrifugation yielded 1.2 +/- 0.4 X 10(6) cells/g lung in the type II cell fractions. The activities of some enzymes involved in the metabolism of xenobiotics were determined in these freshly isolated type II cells and compared with activities in alveolar macrophages and fractions of unseparated cells from the same tissue samples. Reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase activity was similar in the three cell fractions from all five patients (18-29 nmol/mg protein/min). An antibody to rabbit reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase inhibited reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reduction as much as 70% in microsomal preparations of the isolated human pulmonary cells, although this same antibody barely reacted with microsomes of the human cells in a Western blot assay. Epoxide hydrolase activity was highest in the alveolar type II cells (1.08 +/- 0.17 nmol/mg protein/min). This activity was 6 times higher than in the alveolar macrophage or unseparated cell fractions. 7-Ethoxycoumarin deethylase activity, a cytochrome P-450-dependent pathway, was low or undetectable in the three cell fractions. Trace amounts of 7-ethoxyresorufin O-deethylase activity (0.5-1.5 pmol/mg protein/min) were detected in microsomes of the isolated human cells, even though a polycyclic hydrocarbon-inducible cytochrome P-450 which metabolizes 7-ethoxyresorufin (form 6 in rabbits) was not detected immunochemically.
A Percoll density gradient was employed for selecting large numbers of viable insulin-producing RINm5F cells. Homogenates of these cells were then subjected to gradient centrifugation and two clearly visible bands were obtained. The light fraction was essentially composed of mitochondria banded at a density of about 1.06 g/ml. The heavier fraction banded at 1.09 to 1.10 g/ml and contained lysosomes and a small number of secretory granules. The distribution of Percoll particles was restricted to the extracellular space and there was no adsorption to any membrane structures. The distribution pattern of marker enzymes for the mitochondria and lysosomes was similar to that of normal pancreatic beta-cells. With the use of a Percoll density gradient it was thus possible to isolate a purified mitochondrial fraction from viable RINm5F cells.
Isopycnic banding by density gradient centrifugation was used to measure density changes in complexes formed by the interaction between antigen and antibody immobilized on polystyrene latex beads (diameter, 0.109 +/- 0.0025 micron). Measurements of density changes allowed calculation of the interacting masses under the given experimental conditions. Interaction equilibrium constants and free energy change for two sets of reactions, bovine IgG and anti-bovine IgG (rabbit) IgG and rabbit IgG and anti-rabbit IgG (goat) IgG systems, were calculated from isopycnic banding density gradient centrifugation runs. The procedure demonstrates a new method of obtaining quantitative information on antigen-antibody interactions.
Isopycnic banding by density gradient centrifugation was used to measure density changes in complexes formed by the immobilization of each of four different immunoglobulins (IgG) (bovine, dog, rabbit, and sheep) on polystyrene latex beads (0.109 +/- 0.0025 micrometer diameter). Subtractive measurements of density changes allowed calculation of the mass of immobilized IgG under varying experimental conditions. The immobilization data were correlated with adsorption isotherms which incorporated charge repulsion forces. The effects of pH and NaCl concentration on the immobilization were studied for the latex-bovine IgG system. It was found that the mass of immobilized immunoglobulins was increased from 10 to 20% by removing the IgG from its isoelectric range.
A simple procedure for coating the interior surface of Beckman Ultra-Clear centrifuge tubes with polyvinyl alcohol is described. The coated tubes are wettable and allow salt solutions to gravity-feed down their sides. This modification of the tubes permits the performance of previously developed density gradient and uniform density ultracentrifugation procedures for lipoprotein fractionation.
A method is described for the inexpensive and easy preparation of colloidal silica--aluminum modified--polyvinylpyrrolidone (CS-AM-PVP) density gradient centrifugation medium. Using density gradient centrifugation, several cell separation and biochemical characteristics were studied: centrifuge tube wall cell adherence, mouse spleen cell density distribution, rebanding properties, mitogen response and cell aggregation. Cell adherence to the centrifuge tube wall using density gradients of CS-AM-PVP was compared with density gradients of bovine serum albumin and Ficoll. Few cells adhered to the centrifuge tube wall when CS-AM-PVP was used as a gradient medium; whereas, significant cell adherence to the centrifuge tube cell wall occurred when bovine serum albumin or Ficoll was used as a gradient medium. The CS-AM-PVP gradient medium did not inhibit the response to mitogens of mouse spleen cells which had been separated into density subpopulations in a discontinuous CS-AM-PVP density gradient, caused a minimum amount of cell aggregation, and was found to be non-toxic.
The aim of the study was to compare the new density gradient centrifugation system OncoQuick with the standard density gradient centrifugation system Ficoll for improved tumor cell enrichment in blood of tumor patients. Evaluation of OncoQuick and Ficoll density gradient centrifugation was performed by flow-cytometry and immunocytochemistry using 10 ml unspiked and tumor cell-spiked blood samples of tumor-free probands. From 10 ml blood, OncoQuick density gradient centrifugation separated a cell fraction which consisted of a mean cell number of 9.5x10(4) mononuclear cells compared to 1.8x10(7) cells by Ficoll. Density gradient centrifugation of tumor cell-spiked blood samples with OncoQuick and Ficoll led to similar tumor cell recovery rates, between 70% and 90% for both methods. The improved depletion of mononuclear blood cells by OncoQuick simplified further immunocytochemical evaluation of the enriched cell fraction, which could be spun onto 1-2 glass slides by cytocentrifugation. In comparison, the mononuclear cells separated by Ficoll had to be spun onto more than 50 glass slides for complete immunocytochemical evaluation. Consequently, tumor cell density on each cytospin was higher after OncoQuick preparation compared to Ficoll. Density gradient centrifugation with OncoQuick results in higher relative tumor cell enrichment than Ficoll density gradient centrifugation. This simplifies further immunocytochemical tumor cell detection and is a promising tool for the detection of circulating tumor cells in blood of tumor patients.
The fractionation of calf thymus DNA by centrifugation in density gradients of Cs2SO4/BAMD, where BAMD = 3,6-bis(acetatomercurimethyl)dioxane, is described. A large-scale separation of (dG+dC)-rich DNA fractions has been obtained, allowing the relative amounts of minor and satellite components in the bovine genome to be precisely assessed.
The effects of overloading of the sample zone in density gradient centrifugation have been studied by use of a three-component shelf-lavered sample in which the total protein concentration was increased by addition of different amounts of albumin. It is found that overloading of the gradient gives rise to particle movements which are not predictable from the Svedberg equation. The two typical effects of overloading are dislocation of the zone mass centres and changes in the zone shapes. It is found that the magnitude of the calculated sedimentation coefficients increases nearly linearly with increasing sample load. The changes in zone shapes are found to depend on the specific load and two different patterns may be distinguished. The zone of the sample component which causes the overloading is defined as primarily overloaded and the others as secondarily overloaded. In primarily overloaded zones the original Gaussian shape is lost, while in secondarily overloaded zones the Gaussian zone shape is maintained, although a zone broadening is seen. Extreme high loads are found to be able to divide single zones. As a whole these experiments show that evidence for a non-overloaded set of experimental conditions must be provided, when density gradient centrifugation is used for determination of sedimentation coefficients. For preparative gradient centrifugations the power of resolution will decrease with increasing sample load. A simple method to detect overloading in density gradient centrifugations is described.
The use of Percoll for isolation and subfractionation of PBMC and T-lymphocytes by discontinuous and continuous density gradient centrifugation is described: PBMC were isolated from human peripheral blood by discontinuous density gradient centrifugation on Percoll. The use of Percoll instead of Ficoll-Isopaque has the advantage that Percoll, in contrast to Ficoll-Isopaque, does not alter the density of monocytes. Therefore, a better separation of lymphocytes and monocytes was achieved after subsequent continuous density gradient centrifugation on Percoll. E-RFC were isolated by discontinuous density gradient centrifugation after a first low speed centrifugation step banding lymphocytes and SRBC on a Percoll-Ficoll cushion, and a subsequent high speed centrifugation step separating high density rosettes and SRBC from low density non-E-RFC. The advantage of this procedure is the short time of performance and that there is no need to resuspend the lymphocyte/SRBC pellet. PBMC, nph.PBMC T-lymphocytes were further subfractionated by continuous density gradient centrifugation on Percoll. The method described here resulted in a good separation of lymphocytes and monocytes. However, to obtain lymphocyte fractions with minute numbers of contaminating monocytes, a depletion of monocytes prior to further subfractionation of the lymphocytes by continuous density gradient centrifugation is recommended. A marker analysis of T-lymphocytes subfractionated by continuous density gradient centrifugation on Percoll shows that high density T-lymphocytes are enriched in ANAE positive lymphocytes of type 1 and depleted of ANAE positive lymphocytes of type 2. Low density T-lymphocytes are enriched in ANAE type 2 cells and depleted of ANAE type 1 cells. On the other hand, no considerable differences were found when analyzing the T-cells from different fractions for differentiation antigens by means of monoclonal antibodies (anti Lyt 3, OKT4, and OKT8). The results may indicate that subfractionation of T-lymphocytes by continuous density gradient centrifugation on Percoll provided T-cells in different functional states rather than T-cells of distinct subclasses.
Rubella virus was centrifuged in sucrose density gradients. One of two densities could be ascribed to the virus, depending upon the suspending medium used. The virus was found at a density of 1.16 g/cm(3) after centrifugation for 18 hr in sucrose gradients prepared in distilled water. By contrast, when the sucrose gradients were prepared in tris(hydroxymethyl)aminomethane (Tris)buffer containing ethylenediaminetetraacetic acid (EDTA), the virus was found at a density of 1.18 g/cm(3) after 18 hr of centrifugation. The virus banded at this higher density after only 2 hr of centrifugation when pretreated by overnight incubation in the Tris-EDTA buffer. A kinetic study showed that, in sucrose gradients containing this buffer, the virus gradually migrated as a single peak of infectivity from a density of 1.16 g/cm(3) after 2 hr of centrifugation to the higher 1.18 g/cm(3) density after 18 hr. The density change was shown to be reversible; after the removal of the Tris-EDTA buffer, rebanding of virus harvested at the heavy density resulted in its banding at the lower 1.16 g/cm(3) density. The data indicate that density change could not be explained on the basis of the loss of some component from the virus or on the basis of the failure of the virus to reach equilibrium. However, it is possible that the two densities observed were a reflection of the existence of rubella virus in different hydration states in the presence and absence of Tris buffer containing EDTA.
Lymphocytic choriomeningitis (LCM) virus, Traub strain, was purified from BHK-21 tissue culture medium. The virus was then analyzed by equilibrium centrifugation and rate zonal centrifugation in sucrose gradients. A buoyant density in sucrose of 1.18 g/ml was found and the S(20, w) value was estimated to be about 470 to 500S. Furthermore, the (3)H-uridine-labeled ribonucleic acid (RNA) from virus was extracted from LCM virus and analyzed by rate zonal centrifugation. Two major and one minor single-stranded RNA components were found with sedimentation coefficients of 28, 22, and 18S.
In patients with marked hypereosinophilia 'hypodense' and 'normodense' eosinophils have been found after density gradient centrifugation. Subsequently this terminology has also been used in studies of patients with milder eosinophilia. However, in these cases the differentiation between normo- and hypodense eosinophils was less clear. This might be due to the high imprecision of the test of density gradient centrifugation, as demonstrated in the first part of this study: the mean within-assay variance of the number of eosinophils in the different density layers was 35%. It was calculated that the test must be performed eight times to obtain an estimate of the true mean for the individual patient. In the second part of the study, the absolute number of 'hypodense eosinophils' in groups of patients with asthma (adults and children) and rheumatoid arthritis (adults) were compared to normal controls. Although a difference in the absolute number of hypodense eosinophils between groups of patients and controls could be demonstrated, the high imprecision of the test of density gradient centrifugation suggested that the technique used was not useful in an individual with normal or slightly elevated eosinophils in the peripheral blood.
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Bronchopulmonary lavage was performed in 10 healthy horses and in 39 horses with chronic pulmonary disease. The predominant cell types were macrophages in healthy horses and neutrophils in severely diseased horses. Procoagulant activity (PCA) was detected in all 32 cell-free supernatants examined and in all 49 unpurified cell suspensions. Cells were separated by centrifugation on discontinuous gradients prepared either with Percoll or with Metrizamide. Macrophages were enriched in subpopulations of low density. Neutrophils could not be purified by density gradient centrifugation using either gradient medium. PCAs of cell subpopulations were plotted against their respective macrophage, neutrophil, and lymphocyte content. PCA was positively correlated with macrophage content (P less than 0.001) and negatively correlated with neutrophil (P less than 0.02) and lymphocyte (P less than 0.001) content. Therefore, PCA of equine lung cells most likely originates from macrophages as shown in other species. The density shift of lung neutrophils requires further investigation.
An efficient method for the isolation of RNA from cartilage is described. The difficulties in obtaining RNA from cartilage, a tissue of low cell density and high proteoglycan content, were overcome by making several modifications to the guanidine thiocyanate/cesium chloride method of RNA extraction. Cartilage tissue is frozen, crushed, and homogenized in a 4 M guanidine thiocyanate lysis buffer. The RNA is then pelleted by ultracentrifugation through a cesium trifluoroacetate density gradient. The use of cesium trifluoroacetate, rather than cesium chloride, for density gradient centrifugation improves both the yield and purity of total RNA isolated from cartilage. The ultracentrifugation has been adapted to the Beckman TL100 tabletop centrifuge and is complete in 3 h. This fast, simple method produces high quality RNA, suitable for use in RNase protection assays, polymerase chain reaction analysis, and Northern analysis. This purification procedure may be applicable to other sources, from which RNA isolation is complicated by the presence of abundant cell wall or matrix components.