Density Gradient Centrifugation of Rubella Virus

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/cm³ 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/cm³ 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/cm³ after 2 hr of centrifugation to the higher 1.18 g/cm³ 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/cm³ 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.     

Purification of Poly-beta-Hydroxybutyrate by Density Gradient Centrifugation in Sodium Bromide

Fractionation of fully sporulated cultures of Bacillus thuringiensis by density gradient centrifugation in NaBr produced two bands which were identified as poly-beta-hydroxybutyrate. This technique generated high yields of membrane-bound and unbound granules of exceptional purity and degree of polymerization.     

Fractionation of Eastern Equine Encephalitis Virus by Density Gradient Centrifugation in CsCl

When partially purified Eastern equine encephalitis (EEE) virus was centrifuged to equilibrium in CsCl, three virus specific bands were observed. A hemagglutinin was detected at a buoyant density of 1.18 g/cm³. Infectious EEE virus banded in two positions; most of the virus banded at 1.20 g/cm³ and a lesser amount banded at 1.22 to 1.23 g/cm³. Analysis of radioactive profiles of CsCl-fractionated EEE virus labeled with either ³²PO₄ or ³H-uridine suggested that the hemagglutinin was stripped from the intact EEE virion. The viral origin of the hemagglutinin was verified by inhibition with specific antiserum. Attempts to differentiate between infectious EEE virus of the different buoyant densities showed that the denser particle was neither a virus contaminant nor a density mutant. No evidence was obtained to indicate that the denser particle was an immature form of EEE virus. The two infectious EEE species obtained after CsCl fractionation were indistinguishable antigenically. Furthermore, unfractionated as well as CsCl-fractionated EEE virus sedimented at about 260S in sucrose gradients. These results together with the results of rebanding experiments suggested that the denser EEE species (1.23 g/cm³) results from a salt (CsCl)-induced alteration or breakdown of the EEE virion (1.20 g/cm³), and that it arises as the hemagglutinin is stripped from the surface of the EEE virion.     

Large-Scale Purification of Ribonucleic Acid Tumor Viruses by Use of Continuous-Flow Density Gradient Centrifugation

This report describes a 200- to 300-fold increase in the quantity of ribonucleic acud tumor virus particles previously isolated at one time by zonal centrifugation.     

Purification of Poly-β-Hydroxybutyrate by Density Gradient Centrifugation in Sodium Bromide

Fractionation of fully sporulated cultures of Bacillus thuringiensis by density gradient centrifugation in NaBr produced two bands which were identified as poly-β-hydroxybutyrate. This technique generated high yields of membrane-bound and unbound granules of exceptional purity and degree of polymerization.     

密度梯度离心分离大鼠卵巢卵泡膜细胞

探讨用密度梯度离心法快速、有效地分离大鼠卵泡膜细胞.选取23～25 d雌性大鼠卵巢,用Percoll密度梯度离心法将卵泡膜细胞分离纯化,3β-羟基类固醇脱氢酶(3β-hydroxysteroid dehydrogenase,3β-HSD)组织化学染色用于卵泡膜细胞纯度检测.分别用0.1 U/mL和1.0 U/mL卵泡刺激素(Follicle-stimulating hormone,FSH)及黄体生成素(luteinizing hormone,LH)处理细胞,无血清培养48 h后,酶联免疫法检测培养液中雄烯二酮和雌二醇的水平.分离所得细胞中,3β-HSD染色阳性细胞与总细胞数之比大于90％; LH组的雄烯二酮水平显著高于对照组和FSH组(P＜0.05),LH组中1.0 U/mL组的雄烯二酮水平又高于0.1 U/mL组.各组均未检测到雌二醇及孕酮.3β-HSD组织化学染色可快速有效地检验所分离的卵泡膜细胞的纯度,分离所得的卵泡膜细胞可对LH产生反应,且其中几乎没有混杂颗粒细胞.     

CsCl Density Gradient Centrifugation Studies of Intact Bacterial Cells

Cells of Escherichia coli have been successfully banded in CsCl density gradients and a portion of the population reclaimed in a viable state. Differentiation between two strains of this organism in a CsCl density gradient has been demonstrated also. Several studies were undertaken to see whether differences could be detected between two samples of cells of the same strain which had been subjected to different conditions. The results were as follows: (a) Introduction of a heavy label (5-bromouracil) into the DNA during a 90 minute period did not produce an observable change in cell density. (b) Removal of a required amino acid from the growth medium of an E. coli auxotroph resulted in an increase in both the density and heterogeneity of the cells. (c) Exposure of cells to 27 kr of gamma radiation, followed by a period during which portions of both DNA and RNA were lost, yielded two distinct bands, one at the normal position in the gradient and the other shifted to a lighter region.     

Separation of Non-filamentous Micro-organisms from Soil by Density Gradient Centrifugation in Percoll

Soil suspensions were homogenized, and desorbed non-filamentous micro-organisms were concentrated in a minimum volume of buffer by low speed centrifugation. The cells were separated from inanimate material by flotation at the interface between the buffer and a silica sol/polyvinyl pyrrolidone density gradient medium (Percoll). Cell suspensions were removed from the interface and fractionated according to density by high speed centrifugation on discriminating density gradients in Percoll.
Preliminary experiments indicated that most non-filamentous soil micro-organisms had densities in the range 1.081–1.123 g%sol;ml while Rhizobium isolated from crushed root nodules on Percoll was split into two bands of densities 1.081–1.110 and 1.041–1.073 g/ml. The lighter cells were the more pleomorphic.
The efficiency of extraction of cells from soil was governed by the extent of their desorption from inanimate particles. As rigorous desorption procedures damage cells, extraction efficiencies were low; 10–20% of cells counted microscopically in soil were recovered from density gradients. Electron microscopy of soil micro-organisms isolated by this method showed an unusual range of surface ornamentations on cell-like structures of bacterial dimensions.     

Density Gradient Centrifugation Compromises Bone Marrow Mononuclear Cell Yield

Bone marrow mononuclear cells (BMNCs) are widely used in regenerative medicine, but recent data suggests that the isolation of BMNCs by commonly used Ficoll-Paque density gradient centrifugation (DGC) causes significant cell loss and influences graft function. The objective of this study was to determine in an animal study whether and how Ficoll-Paque DGC affects the yield and composition of BMNCs compared to alternative isolation methods such as adjusted Percoll DGC or immunomagnetic separation of polymorphonuclear cells (PMNs). Each isolation procedure was confounded by a significant loss of BMNCs that was maximal after Ficoll-Paque DGC, moderate after adjusted Percoll DGC and least after immunomagnetic PMN depletion (25.6±5.8%, 51.5±2.3 and 72.3±6.7% recovery of total BMNCs in lysed bone marrow). Interestingly, proportions of BMNC subpopulations resembled those of lysed bone marrow indicating symmetric BMNC loss independent from the isolation protocol. Hematopoietic stem cell (HSC) content, determined by colony-forming units for granulocytes-macrophages (CFU-GM), was significantly reduced after Ficoll-Paque DGC compared to Percoll DGC and immunomagnetic PMN depletion. Finally, in a proof-of-concept study, we successfully applied the protocol for BMNC isolation by immunodepletion to fresh human bone marrow aspirates. Our findings indicate that the common method to isolate BMNCs in both preclinical and clinical research can be considerably improved by replacing Ficoll-Paque DGC with adapted Percoll DGC, or particularly by immunodepletion of PMNs.     

Separation of Particulate Phytochrome and Plasma Membranes of Maize Coleoptiles by Density Gradient Centrifugation

Phytochrome, cross linked in situ to its receptor by glutaraldehydefixation, and radioactively-labelled membrane material, obtainedby lactoperoxidase-catalysed ¹²⁵I-treatment of maize coleoptilescould be separated from each other according to sedimentationvelocity and by sucrose density gradient centrifugation. Bindingof iodine to membrane material in maize coleoptiles increasedseveral-fold on the addition of reaction-specific enzymes, i.e.lactoperoxidase and glucose oxidase. Mitochondria were considerednot labelled because mito-chondrial purification reduced ¹²⁵Iincorporation. Membrane material containing incorporated iodineappeared quite heterogenous and sedimented to an equilibriumdensity position close to, but slightly lighter than that ofthe mitochondria; participate phytochrome banded at a heavierposition. Results obtained therefore suggest that the receptorfor phytochrome may not be on the plasma membrane as envisagedin recent hypotheses.     

Improvement of Transfection Efficiency in Cultured Chicken Primordial Germ Cells by Percoll Density Gradient Centrifugation

Chicken primordial germ cells (PGCs) differentiate into germ cells in gonads. Because PGCs can be cloned and cultured maintaining germline competency, they are a good means of modifing the chicken genome, but the efficiency of plasmid transfection into PGCs is very low. In this study, I attempted to improve the efficiency of PGC transfection. Cultured PGCs were purified by Percoll density gradient centrifugation, and were then transfected with plasmid DNA. For transient transfection, the transfection efficiency increased more than 7-fold by the Percoll method. The efficiency of stable transfection of PGCs also increased significantly. The stable transfectants that were isolated by this method accumulated in the developing gonads after microinjection into bloodstream of chick embryos, indicating that gene transfection by Percoll purification did not alter the function of PGCs in vivo.     

Membrane Populations of Bovine Choroid Plexus: Separation by Density Gradient Centrifugation in Modified Colloidal Silica

Abstract: The choroid plexus is intimately involved in the production and regulation of the cerebrospinal fluid. Populations of surface membranes from this epithelial tissue were separated by density gradient centrifugation by use of modified colloidal silica (Percoll). A fraction of heavy microsomes (P₃) containing plasma membranes was prepared by differential centrifugation. Membranes in fraction P₃ were mixed with a given concentration of Percoll and density gradients generated during centrifugation. When fraction P₃ was mixed with 20% (v/v) Percoll and centrifuged at 20,000 r.p.m. for 1 h in a 50.2 Ti fixed-angle rotor, membranes containing alkaline phosphatase (AP) were found at a density of 1.037 g/cm³ while those containing NaK ATPase were found at 1.047 g/cm³. With more shallow density gradients using 12% and 14% Percoll, a broad shoulder of AP activity became manifest at densities greater than 1.060 g/cm³ suggesting multiple populations of membranes containing AP. Membranes containing AP could also be separated from membranes containing γ-glutamyl transpeptidase (γ-GTP); this separation was most pronounced in 12% Percoll. The activity of γ-GTP could not be separated from activity of NaK ATPase. Total protein was distributed broadly throughout the gradients. Studies have been undertaken to compare the behavior of choroidal membranes in Percoll gradients with that of renal membranes because the biochemical anatomy of the kidney has been extensively studied. In contrast to choroidal membranes, renal membranes with NaK ATPase activity were found to have densities lower than those membranes with AP. Thus, the distribution of membrane-bound enzymes from kidney in a Percoll gradient was exactly the opposite of that observed for these same enzymes from choroid plexus. In addition, unlike the γ-GTP activity of choroid plexus, γ-GTP from kidney could be separated from the activities of both alkaline phosphatase and NaK ATPase. These marked differences in membrane populations between choroid plexus and kidney as defined by Percoll density gradient centrifugation analyses are presumably reflective of differences in the functions of the two epithelial tissues.     

Fractionation of Isolated Rat CNS Myelinated Axons by Sucrose Density Gradient Centrifugation in a Zonal Rotor

Myelinated axons isolated from rat CNS brain stem by flotation in a buffered sucrose-salt medium were shocked by vigorous homogenization in hypotonie buffer and then fractionated on a 20-40% (wt/wt) linear sucrose gradient in a Beckman Ti-14 Zonal Rotor. After centrifu-gation to equilibrium, the gradient was fractionated on the basis of sucrose density into 13 individual fractions. The distributions of molecular markers related to myelin [(myelin basic protein, 2’3′-cyclic nucleotide 3′-phos-phodiesterase (EC 3.1.4.37), myelin-associated glycopro-tein (MAG)]; microsomes [CDP-choline:l,2 diglyceride cholinephosphotransferase (EC 2.7.8.2)]; mitochondria [cytochrome c oxidase (EC 1.9.3.1), monoamine oxidase (amine:oxygen oxidoreductase, deaminating, EC 1.4.3.4)], and axolemma [acetylcholinesterase (acetylcho-line hydrolase, EC 3.1.1.7), 5′-nucleotidase (5′-ribonu-cleotide phosphohydrolase, EC 3.1.3.5), Na⁺,K⁺-adeno-sine triphosphatase (EC 3.6.1.3), [³H]saxitoxin binding] were examined, as well as the protein composition and morphological appearance of the fractions. The myelin-related markers were most enriched in the 20-26% region of the gradient, although the MAG was broadly distributed throughout the entire gradient. The axolemma-related markers were most enriched in the 28-32% region of the gradient, whereas the microsomal and mitochondrial-related markers were enriched in the 35-40% region of the sucrose density gradient. Mixing experiments utilizing ¹²⁵I-labeled membrane preparations derived from cultured oligodendroglial and astroglial cells indicated that the constituents of the shocked myelinated axons were not significantly contaminated with glial membranes. The morphology of the fraction was consistent with the membrane molecular marker distribution: the light end of the gradient contained multilamellar myelin; fractions in the center of the gradient were enriched in un-ilamellar membrane fragments; the densest regions of the gradient were enriched in mitochondria. The myelin specific proteins were the prominent polypeptides in the 20-25% regions of the gradient, whereas polypeptides having a molecular weight of 50,000 or greater predominanted in the denser regions of the gradient. The significance of the distribution of these membrane markers and the utility of this fractionation procedure are discussed.     

大规模区带离心纯化Vero细胞乙脑疫苗

本文报道一种适合疫苗生产的大规模纯化Ｖｅｒｏ细胞乙脑疫苗的方法。原疫苗经适当浓缩和去除ＤＮＡ处理后,用不连续蔗糖梯度（３６％和６０％）。３２６００ｇ,速率区带离心４ｈ。纯化后疫苗的效力比中国参考疫苗高出６倍以上,补体结合抗原比中国参考疫苗高４～８倍。总蛋白含量低于３０μｇ／ｍＬ,牛血清含量降至０．５μｇ／ｍＬ以下,细胞残余ＤＮＡ低于１００ｐｇ／０．５ｍＬ。用此法连续制备三批纯化疫苗,其纯度和效力均高于日本鼠脑纯化疫苗。此法对于制备其它纯化Ｖｅｒｏ细胞疫苗也具有一定的参考意义。     

Recovery of Murine Leukemia Virus from Large Volumes of Freshly Harvested Culture Fluids by Using a Single Density Gradient

Concentration and purification of murine leukemia virus for use as complement fixation antigens was accomplished by using a single density gradient in the model RK ultracentrifuge.     

The Use of Density Gradient Centrifugation for the Separation of Germinated from Nongerminated Spores

S ummary : The density gradient centrifugation of a suspension of spores of B. subtilis 8057 on both sucrose and renografin gradients gave 2 distinct fractions. Germination evidence suggested that the heavier fraction consisted of dormant spores and the less dense fraction, germinated spores. It is concluded that density gradient centrifugation may provide a useful technique for the separation of germinated from nongerminated spores.     

Purification and Aggregation of Influenza Virus by Precipitation with Polyethylene Glycol

Influenza virus may be purified and rendered free of extraneous proteins by precipitation and aggregation with polyethylene glycol at polymer concentrations of 1 to 4%. The precipitated virus is superior antigenically to the virus in monomeric and in the ether dissociated forms. When the virus is precipitated at polyethylene glycol concentrations of 5% and higher the virus is not aggregated and is associated with extraneous protein which co-precipitates with the infectious agent.     

Separation of Viable Rickettsia typhi from Yolk Sac and L Cell Host Components by Renografin Density Gradient Centrifugation

Rickettsia typhi cultivated in the yolk sac of chicken embryos or in L cells irradiated 7 days previously was separated from host cell components by two cycles of Renografin density gradient centrifugation. Preliminary steps involved differential centrifugation and centrifugation over a layer of 10% bovine plasma albumin of infected yolk sac suspensions, or trypsinization and passage through filters of wide porosity of infected L cell suspensions. Rickettsial preparations obtained by these methods appeared to be free from host cell components while retaining high levels of hemolytic activity, egg infectivity, and capacity to catabolize glutamate. Average yields were 3.3 mg of rickettsial protein per yolk sac or 0.44 mg per 16-oz (ca. 475-ml) L cell culture. Extracts from these two preparations displayed malate dehydrogenase activity of electrophoretic mobility identical to each other but quite different in migration patterns from the corresponding host cell enzymes. This method of separation of rickettsiae from host cell constituents appears to be particularly well suited for the study of rickettsial enzymatic activity.     

Monitoring Microbial Numbers in Food by Density Centrifugation

Some foods contain low numbers of microbes that may be difficult to enumerate by the plate count method due to small food particles that interfere with the counting of colonies. Ludox colloidal silicon was coated with reducing agents to produce a nontoxic density material. Food homogenates were applied to a layered 10 and 80% mixture of modified Ludox and centrifuged at low speed. The top and bottom of the tube contained the food material, and the Ludox-containing portion was evaluated by conventional pour plate techniques. Plate counts of the Ludox mixture agreed with plate counts of the food homogenate alone. The absence of small food particles from pour plates resulted in a plate that was more easily read than pour plates of the homogenate alone. Modified Ludox was evaluated for its effect on bacteria at 4°C during a 24-h incubation period. No inhibition was observed. This method is applicable to food products, such as doughnuts, spices, tomato products, and meat, in which small food particles often interfere with routine plate counts or low dilution may inhibit colony formation. Inhibitory substances can be removed from spices, resulting in higher counts. Ludox is more economical than similar products, such as Percoll. Modified Ludox is easily rendered nontoxic by the addition of common laboratory reagents. In addition, the mixture is compatible with microbiological media.