Toxoplasma gondii: large-scale purification by zonal density gradient centrifugation.

This study shows the feasibility of using density gradient centrifugation in the “A” zonal rotor for large-scale purification of Toxoplasma gondii tachyzoites from the host cells of mouse peritoneal exudate. Ficoll and Dextran 40 were used as gradients. Using Ficoll gradient, up to 70% of the toxoplasms were recovered by pooling purified fractions with the peak fractions giving recoveries around 38%. In the experiment using dextran gradients Toxoplasma recovery was of 41%, but the band of host cells was not so sharply formed.     

Isolation of anucleated yeast protoplasts by means of density gradient centrifugation

An improved method for the preparation in high yield of anucleated Saccharomyces cerevisiae has been developed. This method is based on a two-stage centrifugation of the original protoplast mixture in linear density gradients (1–10%, w/v) of Ficoll 400. The yield of anucleated protoplasts was 5–9%, its value depended on the frequency of the nucleus-free protoplasts in the original mixture.The anucleated protoplasts were characterized by RNA, DNA and protein content, and by light and electron microscopy. The protoplasts lacking nuclei had about one third the diameter of the nucleated ones, and reduced of DNA, RNA and protein in comparison to normal protoplasts. Electron microscopy showed a typical yeast ultrastructure in anucleated protoplasts except that they lacked nuclei and exhibited a higher frequency of lipid granules and exocytotic electron-dense vesicles located close to the plasmalemma.     

Selection of synchronous Chlorella cells by centrifugation to equilibrium in aqueous Ficoll

Synchronous daughter cells of Chlorella pyrenoidosa can be selected, on the basis of their unique density, from cultures growing in continuous light by centrifugation to equilibrium in aqueous Ficoll. The degree of synchrony, as judged from cell release data, is equivalent to or better than that obtained by intermittent-illumination procedures.     

Percoll and Ficoll self-generated density gradients by low-speed osmocentrifugation

Percoll and Ficoll self-generated density gradients can be obtained by low-speed centrifugation of their solutions within dialysis cells. Useful Percoll density gradients can be obtained after 10-30 min centrifugation at 220-2010g, within dialysis cells. Ficoll density gradients, which are more difficult to self-generate, can be obtained by the same technique. Red cell band formation in a Percoll density gradient can be done in a single step by using dialysis cells as the centrifugation solution container.     

Isolation of plasma membranes from corn roots by sucrose density gradient centrifugation: an anomalous effect of ficoll

An investigation was conducted into the isolation of plasma membrane vesicles from primary roots of corn (Zea mays L., WF9 × M14) by sucrose density gradient centrifugation. Identification of plasma membranes in cell fractions was by specific staining with the periodic-chromic-phosphotungstic acid procedure. Plasma membrane vesicles were rich in K⁺-stimulated ATPase activity at pH 6.5, and equilibrated in linear gradients of sucrose at a peak density of about 1.165 g/cc. It was necessary to remove mitochondria (equilibrium density of 1.18 g/cc) from the homogenate before density gradient centrifugation to minimize mitochondrial contamination of the plasma membrane fraction. Endoplasmic reticulum (NADH-cytochrome c reductase) and Golgi apparatus (latent IDPase) had equilibrium densities in sucrose of about 1.10 g/cc and 1.12 to 1.15 g/cc, respectively. A correlation (r = 0.975) was observed between K⁺-stimulated ATPase activity at pH 6.5 and the content of plasma membranes in various cell fractions. ATPase activity at pH 9 and cytochrome c oxidase activity were also correlated.     

Buoyant density constancy during the cell cycle of Escherichia coli

Cell buoyant densities were determined in exponentially growing cultures of Escherichia coli B/r NC32 and E. coli K-12 PAT84 by equilibrium centrifugation in Percoll gradients. Distributions within density bands were measured as viable cells or total numbers of cells. At all growth rates, buoyant densities had narrow normal distributions with essentially the same value for the coefficient of variation, 0.15%. When the density distributions were determined in Ficoll gradients, they were more than twice as broad, but this increased variability was associated with the binding of Ficoll to the bacteria. Mean cell volumes and cell lengths were independent of cell densities in Percoll bands, within experimental errors, both in slowly and in rapidly growing cultures. Buoyant densities of cells separated by size, and therefore by age, in sucrose gradients also were observed to be independent of age. The results make unlikely any stepwise change in mean buoyant density of 0.1% or more during the cycle. These results also make it unlikely that signaling functions for cell division or for other cell cycle events are provided by density variations.     

Isolation of Intact Chloroplasts from Euglena gracilis by Zonal Centrifugation

Chloroplasts were separated from Euglena gracilis by zonal centrifugation at low speed in density gradients of Ficoll or dextran. The chloroplasts were intact by the criteria of ultrastructure and their content of ribulose diphosphate carboxylase and soluble protein. The chloroplasts also contained ribosomes and ribosomal RNA uncontaminated by the corresponding cytoplasmic particles.     

Separation of mouse testis cells by equilibrium density centrifugation in Renografin gradients

Mouse testis cells have been separated by equilibrium density centrifugation in gradients of Renografin. Intact testis cells were not damaged by the separation procedure provided that, following separation, the osmolarity was reduced gradually. The various cell types were identified microscopically and by ³H-thymidine labelling with similar results. The present technique has demonstrated that significant variations in cell density occur during spermatogenesis. Approximately ten-fold enrichments of nearly all testis cell types were achieved by equilibrium density separation of testis cell suspensions. More homogeneous cell populations were prepared by density gradient centrifugation of cell fractions obtained from velocity sedimentation separations. Overall enrichments of spermatogonia, by 29-fold; pachytene spermatocytes, 45-fold; dividing meiotic cells, 170-fold; round spermatids, 30-fold; step 11–13 elongating spermatids, 12-fold; Leydig cells, 70-fold; and cytoplasmic fragments, 55-fold, were obtained. In this study, a method for preparation of cell suspensions was also developed to produce higher yields of spermatogonia and young primary spermatocytes; however, the density distribution of these cells was altered.     

Separation of European flat oyster, Ostrea edulis, haemocytes by density gradient centrifugation and SDS-PAGE characterisation of separated haemocyte sub-populations

A two-step gradient centrifugation with Percoll and Ficoll successively as density medium was developed to separate European flat oyster, Ostrea edulis, haemocytes into three sub-populations representing granulocytes, large hyalinocytes and small hyalinocytes, respectively. After a Percoll gradient centrifugation, granulocytes and agranulocytes were separated and a pure fraction of granulocytes was obtained. The agranulocytes were further separated by centrifugation through a Ficoll gradient, and two haemocyte subpopulations representing large hyalinocytes and small hyalinocytes were obtained. No significant impact on the haemocyte viability was detected after separation with this two-step density gradient centrifugation. The three haemocyte sub-populations showed different protein patterns in SDS-PAGE.     

Purification of synaptic vesicles from elasmobranch electric organ and the use of biophysical criteria to demonstrate purity.

We have purified cholinergic synaptic vesicles from the electric organs of two related marine elasmobranchs, Torpedo californica and Narcine brasiliensis, to a specific activity higher than had previously been obtained. We have demonstrated the homogeneity of the vesicles by biophysical criteria. The purification scheme consisted of differential centrifugation, flotation equilibrium in sucrose density gradients, and permeation chromatography on glass bead columns of average pore size 3000 A. Our criteria for purity were that bound acetylcholine, bound nucleotide triphosphate, protein, and lipid--phosphorus behave identically when vesicles were analyzed by procedures which depend on vesicle size, density, and charge. Contaminants were not detected when vesicles were fractionated by preparative and analytical sedimentation, by preparative equilibrium sedimentation using glycerol density gradients, or by electrophoresis in Ficoll density gradients. Pure synaptic vesicles, which have been purified 290-fold from the initial homogenate, contain per mg of protein: 8 mumol of acetylcholine, 3 mumol of ATP, and 7 mumol of lipid phosphorus. These procedures may be of general value in the purification of membrane vesicles.     

Analysis by isopycnic centrifugation of isolated nucleoids of Escherichia coli.

The isolated, formaldehyde-fixed nucleoid of E. coli has been analyzed by isopycnic centrifugation in CsCl density gradients. The membrane-free nucleoid bands at a density of 1.69 +/- 0.02 g/cm3. The membrane-associated nucleoid bands at a density of 1.46 +/- 0.02 g/cm3. Both species sediment to equilibrium as nearly monodisperse bands in CsCl, suggesting that the nucleoid components of DNA, RNA and protein are present in relatively constant ratios. These ratios are constant regardless of the position of the nucleoids in the heterogeneous sedimentation profile of a preparative sucrose gradient. The fixed nucleoids remain condensed during isopycnic centrifugation and there is no detectable loss of RNA from the nucleoid.     

Oxidative phosphorylation in rat liver mitochondria isolated by rate zonal centrifugation: Examination of Ficoll gradients and subpopulations of mitochondria

In order to measure the parameters of oxidative phosphorylation it is necessary to isolate physiologically intact mitochondria. The isolation of rat liver mitochondria by rate zonal centrifugation utilizing isoosmotic Ficoll gradients resulted in the uncoupling of oxidative phosphorylation in these organelles. Analysis of the Ficoll solutions used to construct the gradients indicated that the Ca²⁺ content (200–400 nmole Ca²⁺/mg protein) was sufficiently high to cause an uncoupling of oxidative phosphorylation. Treatment of the Ficoll solutions with Amberlite MB-3 resin reduced the Ca²⁺ content to levels below the limit of determination of the assay procedure. This resulted in the retention of respiratory control (1.42) in rate-zonally centrifuged mitochondria. The addition of bovine serum albumin (100 mg%) to the Ficoll gradients increased the respiratory control index to 2.10. The increase is due to an elevation in state 3 respiration rather than any change in state 4 respiration. The addition of 200 mg% bovine serum albumin to the Ficoll gradient did not further enhance the respiratory control index.Examination of subpopulations of rat liver mitochondria revealed that they are heterogeneous with regard to states 3 and 4 respiration, respiratory control indices, and ADP:O ratios. In mitochondrial subpopulations respiratory control indices ranged from 1.00 to 4.13 and ADP:O ratios ranged from 1.22 to 1.83. This investigation defined a procedure for the isolation of physiologically intact mitochondria from rat liver homogenates.     

Impact of Ficoll density gradient centrifugation on major and trace element concentrations in erythrocytes and blood plasma

ProjectFicoll density gradient centrifugation is widely used to separate cellular components of human blood. We evaluated the suitability to use erythrocytes and blood plasma obtained from Ficoll centrifugation for assessment of elemental concentrations.ProcedureWe determined 22 elements (from Li to U) in erythrocytes and blood plasma separated by direct or Ficoll density gradient centrifugation, using inductively coupled plasma mass spectrometry.ResultsCompared with erythrocytes and blood plasma separated by direct centrifugation, those separated by Ficoll had highly elevated iodine and Ba concentration, due to the contamination from the Ficoll-Paque medium, and about twice as high concentrations of Sr and Mo in erythrocytes. On the other hand, the concentrations of Ca in erythrocytes and plasma were markedly reduced by the Ficoll separation, to some extent also Li, Co, Cu, and U. The reduced concentrations were probably due to EDTA, a chelator present in the Ficoll medium. Arsenic concentrations seemed to be lowered by Ficoll, probably in a species-specific manner. The concentrations of Mg, P, S, K, Fe, Zn, Se, Rb, and Cs were not affected in the erythrocytes, but decreased in plasma. Concentrations of Mn, Cd, and Pb were not affected in erythrocytes, but in plasma affected by EDTA and/or pre-analytical contamination.ConclusionsFicoll separation changed the concentrations of Li, Ca, Co, Cu, As, Mo, I, Ba, and U in erythrocytes and blood plasma, Sr in erythrocytes, and Mg, P, S, K, Fe, Zn, Se, Rb and Cs in blood plasma, to an extent that will invalidate evaluation of deficiencies or excess intakes.     

Large-scale purification of Qbeta-virus by differential centrifugation

Bacterial RNA virus, Qβ, has been purified in gram amounts by differential centrifugation. Final separation of the virus from host E. coli rRNA was based on the density differential in the pellet. The method provided a simplified alternative to the more conventional rate zonal or isopycnic zonal centrifugation techniques.     

Production of Ficoll, Percoll, and albumin gradients by the freeze-thaw method.

Ficoll, sucrose, albumin, and Percoll, a modified colloidal silica centrifugation medium, all form density gradients upon freezing and thawing. The data suggest that any density gradient material will form gradients upon freezing and thawing, provided the material is stable to freezing.     

The morphology of erythroid cells separated by density gradient centrifugation through ficoll

Summary The regenerating blood of geese injected with phenylhydrazine was subjected to large scale, zonal centrifugation through density gradients of Ficoll. In this way, erythroid cells were fractionated according to their respective stages of development. Highly enriched fractions were obtained, containing cells that were well preserved as assessed by both light and electron microscopy. The separated cells exhibited ribosome density and nucleic acid and protein staining patterns typically associated with erythrocyte differentiation. Morphometric analysis of nuclei indicated that despite an apparent net increase in the amount of compact chromatin during development, comparatively little difference existed between the volumes of condensed chromatin present in immature and mature cells. Instead, there was a three fold decrease in nuclear volume between young erythroblasts and reticulocytes, coupled with a concomitant decrease in the volume occupied by dispersed chromatin, RNP and nucleoli. These observations are discussed in relation to molecular changes associated with nuclear differentiation in erythroid cells.Supported by grants from the National Research Council of CanadaWe thank Dr. G. Setterfield for assistance with the EM data and we are grateful to the N.R.C. for use of centrifuges and the zonal rotor     

Separation and subtyping of planarian neoblasts by density-gradient centrifugation and staining

A method has been devised to isolate neoblasts from planarians in high purity and high yield. Specimens of Dugesia polychroa and Dugesia tahitiensis were disintegrated mechanically. After several prepurification steps consisting in sequential filtering through increasingly fine meshes and differential centrifugation, the resulting cell suspension was separated by centrifugation in discontinuous density gradients formed from Percoll solutions. Isosmotic conditions were applied. Two gradients are recommended, one isopycnic four-step gradient (densities 1.03, 1.05, 1.07, 1.09) to obtain one single fraction with a high yield of neoblasts in high purity and one six-step gradient for preparing subfractions of neoblasts somewhat less pure, but in still greater amounts. This latter gradient (densities 1.04, 1.05, 1.06, 1.065, 1.07, 1.09) was run under conditions of rate zonal centrifugation and used for further subtyping of neoblasts by specific staining with azure A – eosin B. A first survey of tentative types based on morphological criteria is given. The viability of neoblasts isolated in the way described was tested in primary cell cultures. In current experiments, 6-week-old cultures had a viability of roughly 50%, with mitoses up to 1 week after the isolation of neoblasts. This revised version was published online in July 2006 with corrections to the Cover Date.     

Density gradient centrifugation of hemopoietic colony-forming cells

Buoyant density distributions of hemopoietic colony-forming units (CFU) from normal mouse marrow were determined by equilibrium density gradient centrifugation in bovine serum albumin (BSA) gradients. The distributions were compared with those obtained for the total population of nucleated cells from normal mouse marrow. The buoyant density distribution for CFU was found to differ from the density distribution for the total nucleated cell population, and the portion of the total cell population with densities much less than the mean value was found to contain up to a 30-fold greater proportion of CFU than an uncentrifuged control. These results provide a preliminary approach to the purification and characterization of normal hemopoietic colony-forming stem cells.     

An evaluation of Ficoll density gradient centrifugation as a method for eliminating microbial contamination and purifying plant protoplasts

Ficoll density gradient centrifugation was used to generate sterile protoplasts from non-sterile Pteridium cultures. The method was found to drastically reduce levels of fungal and bacterial contamination introduced prior to centrifugation whilst concentrating and purifying protoplasts.     

Plasmodium yoelii and Plasmodium berghei: Isolation of infected erythrocytes from blood by colloidal silica gradient centrifugation

Percoll (colloidal silica coated with polyvinylpyrrolidone) and Ficoll (MW 400,000) were used to separate erythrocytes infected with Plasmodium yoelii and Plasmodium berghei from uninfected red blood cells. Samples of blood collected from mice in different phases of malarial infection were overlaid on cushions of 55% Percoll, 20% Ficoll, or 28% Ficoll, respectively, centrifuged, and the interphase layers compared. The best yield of parasitized erythrocytes (PE) was achieved using Percoll when about 95% of the erythrocytes infected by the late developmental forms of the parasites (late trophozoites, schizonts, and gametocytes) were recovered from the gradient interphase, irrespective of the phase of the infection and the number of young erythrocytes in the sample. No alteration of antigenicity (assessed by immunofluorescence) or of osmotic fragility (over the range of 160–460 mOsm) could be detected in PE separated by Percoll or by Ficoll. In addition, parasites separated on Percoll gradients showed no significant ultrastructural changes and retained their normal infectivity to mice. Although both gradient media could be used for the separation of Plasmodium-infected erythrocytes, Percoll presented some advantages over Ficoll. Apart from the better reproducibility of the separation of high yields of very pure PE obtained with Percoll, its lower viscosity allowed easier handling, and lower centrifugal forces were needed to enable the cells to reach their isopycnic positions. Thus, Percoll fulfilled many of the criteria for an ideal density gradient medium. Parasitized erythrocytes were isolated by an easy, reproducible, and inexpensive procedure, and separated cells retained their normal structure, antigenicity, and infectivity.