A separation chamber to sort cells,nuclei, and chromosomes at moderate g forces. II. Studies on velocity sedimentation and equilibrium density centrifugation of mammalian cells

A separation chamber having a surface of 50 cm² and a height of 2 cm is described for the rapid separation of cells and cell organelles at acceleration forces from 10 to 90g. To eliminate wall sedimentation artifacts, the chamber was positioned 20 cm from the rotor axis in a speed-controlled centrifuge. The chamber has flow deflectors for the undisturbed introduction of the sample layer and the gradient; an antivortex cross prevents swirling upon acceleration and deceleration. To illustrate the use of the separation chamber, examples of velocity sedimentation and of equilibrium density centrifugation are given: (i) human monocytes (70% were 90% pure) are separated from lymphocytes in 10 min at 20g; (ii) nonparenchymal rat liver cells are separated in 10 min at 16g in 97% pure endothelial cells and 99% pure Kupffer cells; (iii) equilibrium density centrifugation of human peripheral blood cells at about 90g permits the separation of erythrocytes, monocytes, lymphocytes, neutrophils, eosinophils, and basophils in one run. B cells are separated from T cells. The movement of swinging buckets is analyzed in mathematical terms and a simple method is offered to determine the position of cells in density gradients with the use of a small programmable calculator.     

Plasmid chromosome isolation: An improved batch procedure for large plasmids

A one-step, batch fractionation procedure for the isolation of Escherichia coli plasmids with molecular weights up to at least 1.3 × 10⁸ has been devised. A 15-ml lysate of 5 × 10¹⁰ bacteria is gently prepared and underlain with 15 ml of CsCl-ethidium bromide solution in a 30-ml centrifuge bottle. During an 18- to 24-h 100,000g centrifugation in an anglehead rotor, an aggregate of dense cellular debris including almost all of the host DNA pellets. Plasmid chromosomes are banded in the supernatant isopycnic gradient and are detected by fluorescence of their intercalated ethidium bromide. Plasmid recoveries of 40–70% are achieved. The preparations are pure enough to be used for plasmid circumference measurements by electron microscopy.     

Fractionation of rat fibroblasts in a zonal rotor by means of a viscosity barrier.

Cell fractionation procedures involving differential sedimentation followed by resuspension of pellets and isopycnic centrifugation are very difficult to apply to the small amounts of material available from tissue culture cells. We have explored the possibility of successive differential and isopycnic sedimentation in a zonal rotor using a short viscosity barrier for the differential sedimentation. The marker enzymes used were cytochrome oxidase, acid phosphatase, catalase, and 5′-nucleotidase. The results of these procedures are compared to the results of one-step isopycnic separations in gradients of sucrose and Stractan. The Stractan gradient was much more effective than the sucrose gradient in separating the marker enzymes from the proteins of a postnuclear supernatant, but neither type of gradient could significantly purify the marker enzymes one from another. A two-step procedure using a viscosity barrier was effective in separating particles carrying catalase from the other marker enzymes assayed and from most of the protein. A three-step procedure resulted in similar purification of mitochondria. Modification of barrier composition and centrifugation times would probably result in further improvement of separations according to individual requirements for yield, purification, and freedom from specific contamination by other subcellular particles.     

Purification and some properties of Tephrosia symptomless virus

Tephrosia symptomless virus (TSV), isolated from Tephrosia villosa, is widely distributed in coastal districts of Kenya. The virus was readily transmitted by inoculation of sap, but not by Aphis craccivora or Apion sp. (Curculionidae) or through soil. Host range was very restricted and it infected only 10 of 70 species tested in one of nine plant families; susceptible species were confined to five genera within the Papilionaceae. The virus was cultured, propagated and assayed in soybean. TSV remained infective after 10 min at 85°C, 3 wk at 20°C and 26 wk at -12°C; crude infective sap of Glycine max retained infectivity when diluted 10^-6 but not 10^-7. Virus was purified from systemically infected soybean by clarifying sap extracted in 0.06 m phosphate buffer containing 0.001 m EDTA and 0.1% thioglycollic acid (pH 7.5) with equal volumes of 1:1 n-butanol/chloroform followed by two cycles of differential and one of sucrose density gradient centrifugation. Purified preparations contained c. 33 nm isometric particles. TSV contained RNA and one protein of molecular weight 1.53. 10⁶ and c. 42 000, respectively. Analytical centrifugation indicated a single component with a sedimentation coefficient (s.20, w) of 127 S; in Cs₂SO₄ and CsCl isopycnic gradients a single virus band formed; buoyant density in CsCl was 1.361. TSV was not related serologically to any of 44 viruses in nine plant virus groups but it resembled the tombusviruses and other ungrouped viruses such as carnation mottle in some of its properties.     

Separation of epithelial cells from suspensions of cells from the hamster parotid gland in an isokinetic density gradient of Ficoll in tissue culture medium.

Epithelial cells were separated from suspensions of hamster parotid cells by velocity sedimentation in an isokinetic gradient and by isopycnic sedimentation. Epithelial cells were 48.1 ± 18.0% of the cells in the starting sample suspensions of cells from the disaggregated hamster parotid glands. The purest gradient fractions following velocity sedimentation in a previously described isokinetic gradient contained 98.8 ± 1.8% epithelial cells. The purest fractions obtained from isopycnic sedimentation contained 99.9 ± 0.2% epithelial cells. Purification of parotid epithelial cells by velocity sedimentation in the isokinetic gradient seems preferable to purification using isopycnic centrifugation because a larger proportion of the epithelial cells are obtained in the zone of the gradient which contains highly purified epithelial cells and because velocity sedimentation requires lower centrifugal forces for a shorter period of time.     

Rottboellia yellow mottle virus,a new sobemovirus affecting Rottboellia cochinchinensis (Itch grass) in Nigeria1

A hitherto undescribed virus, termed rottboellia yellow mottle virus (RoYMV), affecting Rottboellia cochinchinensis (syn. R. exaltata, itch grass) at Ibadan, Nigeria, was investigated. RoYMV virions are isometric, c. 29 nm in diameter, and sediment homogeneously at 114s. In isopycnic CsCl gradients, RoYMV virions band sharply at a buoyant density of 1.379 g cm^-3, but in Cs₂SO₄ gradients, virions band at two zones, at the densities of 1.300 g cm^-3 and 1.325 g cm^-3. Treatment with EDTA at pH 8.0 reduced the sedimentation value of RoYMV to c. 87s and rendered it susceptible to proteinase-K, SDS and NaCl. The apparent molecular weight of RoYMV coat protein was c. 27 000. Virions encapsidate a single-stranded RNA of mol. wt 1.4 × 10⁶ Da. Besides R. cochinchinensis, RoYMV was mechanically transmissible only to maize (Zea mays). No reaction occurred when RoYMV was tested against antisera to 44 isometric plant viruses (belonging to 12 groups), including several that affect Gramineae. RoYMV exhibits striking similarities with other viruses belonging to the sobemovirus group, and it is tentatively designated as a new member of the sobemovirus group.     

Respiratory Syncytial Virus Isolation by Combined Continuous Flow-Isopycnic Banding Centrifugation

A new zonal centrifuge rotor (B-IX) which combines continuous sample flow centrifugation with isopycnic banding has been used to isolate and concentrate respiratory syncytial virus from liter volumes of culture fluid. This isolation technique utilizes a sucrose density gradient to trap and isopycnically band the virus particles, and permits recovery of the particles from the rotor in an unaggregated condition.     

Large-scale preparation of plasma membrane vesicles from PC-12 Pheochromocytoma cells and their use in noradrenaline transport studies

Plasma membranes were isolated from rat pheochromocytoma cells (PC-12) grown in spinner culture. The rapid and simple isolation procedure consisted of a differential and isopycnic centrifugation (in a linear sucrose gradient) with the aid of a high capacity fixed angle rotor equipped with siliconized centrifuge tubes. The isolated membranes were closed and osmotically active vesicles (about 0.3 μm in diameter) with a mean intravesicular water space of 1.84 μl / mg protein. In the presence of an inward gradient of sodium chloride and an outward gradient of potassium, [³H]noradrenaline (50 nM) was taken up and accumulated 550-fold (at 31°C). The uptake and accumulation of [³H]noradrenaline was temperature-sensitive and inhibited by the tricyclic antidepressant desipramine. Membrane vesicles isolated from PC-12 cells represent a useful model for the investigation of the molecular mechanism of the neuronal noradrenaline transport system.     

Concentration and purification of two small RNA viruses: mycophage PS-1 and bacteriophage phi6

Two small RNA viruses, mycophage PS-1, and bacteriophage φ6 were concentrated and purified by the sequential use of continuous flow zonal centrifugation, high speed continuous flow sedimentation, and combined rate zonal centrifugation and isopycnic banding. The continuous flow centrifugations were done using the K-II and K-XI rotors while the BXXIX rotor was used for batch rate zonal centrifugation and isopycnic banding. The deseribed procedures are useful for the concentration and purification of virus from large volumes of culture fluid.     

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.     

Tissue-culture fractionation. Zonal centrifugation of Lettrée cells homogenized by glycerol-induced lysis: subfractionation of membranes in sucrose gradients.

1. Lettrée cells were grown intraperitoneally in MF-1 mice. 2. Cells that were loaded with glycerol were swollen in 0.1 M-sucrose and disrupted by Dounce homogenization. 3. Early-passage Lettrée cells were more easily disrupted than late-passage cells by this method, and the former produced larger fragments of plasma membrane. 4. The membranes were fractionated initially in sucrose gradients (on the basis of sedimentation rate) in a BXIV zonal rotor. 5. Fractions from this gradient were further resolved in isopycnic sucrose gradients. 6. Plasma-membrane and endoplasmic-reticulum fractions were recovered in good yield and high purity.     

Sedimentation studies of giant DNA molecules present in unsheared whole-cell lysates of D. melanogaster cells.

We have used band sedimentation in shallow density gradients of CsCl in the preparative centrifuge to analyze the distribution of sedimentation coefficients present in tritium labelled DNA from D. melanogaster cells. The cells were lysed according to the method of Kavenoff and Zimm to preserve very high molecular weight DNA. Sedimentation measurements have been carried out as a function of speed of centrifugation. The resulting distribution functions have been interpreted with the aid of the Zimm-Schumaker equation for the speed dependence of the sedimentation coefficient of very high molecular weight DNA. Low-speed centrifugation (3000 rpm) indicates that DNA molecules from the lysate are evenly distributed over values of S_20,w from 0 to 514S. This distribution is very sensitive to changes in speed of centrifugation and is transformed into a bimodal distribution at 12,080 rpm. Analysis of this transformation allows us to postulate that perhaps 55% of the DNA in the lysate may have molecular weights in excess of 40 × 10⁹ g/mol. Some of these molecules may also possess a variety of configurations including partially replicated branched structures.     

Isolation of radio-iodinated apical and basal-lateral plasma membranes of toad bladder epithelium

Summary The apical and basal-lateral plasma membranes of toad bladder epithelium were radio-iodinated with the glucose-glucose oxidase-lactoperoxidase system. The covalently bound radio-iodine was used as a marker during subcellular fractionation and membrane isolation. Homogenization conditions that ensured rupture of more than 80% of the cells without substantial nuclear damage were defined by Nomarski optics. The nuclei were separated by differential centrifugation and the apical and basal-lateral components were resolved by differential and sucrose density gradient centrifugation. The apical components yielded two radioactive bands that were identified as glycocalyx and plasma membrane labeled with¹²⁵I. The basal-lateral components yielded a heterodisperse pattern made up of at least 3 radioactive bands, but the bulk of the activity of ouabain-sensitive ATPase comigrated with only one of these bands. The mitochondia, identified by assays for cytochrome oxidase and NADH cytochromec reductase activities, were separated from the radio-iodine labeled components by centrifugation in sucrose density gradients under isokinetic conditions. The labeled glycocalyx and the slowly migrating components of basal-lateral labeling were separated from the radio-iodinated membranes by centrifugation at 100,000 × g × 1 hr after removal of the mitochondria by the isokinetic method. The labeled membranes were then subjected to ultracentrifugation in sucrose density gradients under isopycnic conditions; the basal-lateral membranes containing ouabain-sensitive ATP-ase were well resolved from the apical membranes by this method. These results provide a relatively rapid method of attaining partial purification of the apical and basal-lateral plasma membranes of toad bladder epithelium.     

The evaluation of AKR leukemia virus purity: the requirement for both velocity and isopycnic centrifugation methods.

The purity of AKR murine leukemia virus obtained by isopycnic centrifugation was compared with the purity obtained by combining velocity sedimentation and isopycnic centrifugation methods. Evaluation of AKR and Rauscher viral purity by electron microscopy and by analysis of [³H]uridine-labeled viral RNA demonstrated that the velocity centrifugation step is essential for the removal of contaminants banding in the viral density region (1.19 – 1.15 g/ml). For studies requiring relatively pure oncornavirus preparations, a combination of both velocity sedimentation and isopycnic centrifugation steps are suggested. Viral recovery of about 50% was obtained by the method described.     

THE RESOLUTION OF MIXTURES OF VIABLE MAMMALIAN CELLS INTO HOMOGENEOUS FRACTIONS BY ZONAL CENTRIFUGATION

Large-scale separation of mixtures of mammalian cells was obtained with the A-1X zonal centrifuge rotor and density gradients consisting of Ficoll dissolved in modified Eagle's MEM suspension-culture medium. The cells remained viable as tested by plating efficiency or by motility observed with time-lapse photography. Rabbit thymocyte and HeLa cell mixtures were separated with 99 and 89 per cent purity, respectively. Mixtures of thymocytes and suspension-cultured, human acute leukemia cells (Roswell Park strain LKID) were separated with 93 and 91% purity, respectively. HeLa cells were isolated 92% pure from a mixture with horse leukocytes. A book of charts giving the sedimentation position and velocity versus time of cells in the A rotor under standard conditions of gradient composition, angular velocity, and temperature was prepared with the use of a computer program based on the differential sedimentation equation. The charts are used to estimate the centrifugation time necessary for maximum separation of cells. The success achieved in separating mixtures of cells points to the future possibility of large-scale fractionation of solid tissues, especially tumor tissues, into preparations cf viable cells of a single type.     

A Rapid Method of Isolation and Fractionation of Proteoglycans Using a Vertical Tube Rotor for Isopycnic Centrifugation

A comparison was made between a vertical tube rotor and a fixed angle rotor for isopycnic centrifugation of proteoglycans. In the vertical tube rotor, isopycnic gradient was achieved much faster than in the conventional fixed angle rotor. The use of a vertical tube rotor for isopycnic centrifugation shortens the time considerably for the isolation of proteoglycans fron various tissues.     

Analytical subcellular fractionation of rat pituitary homogenates with special reference to the subcellular localization and properties of alkaline phosphatases

Alkaline phosphatase activities of the virgin rat anterior pituitary were studied with a highly sensitive fluorometric assay. Tissue whole homogenates were fractionated on sucrose density gradients in a Beaufay automatic zonal rotor and the gradient fractions assayed for alkaline phosphatase, prolactin and various organelle marker enzymes. Alkaline phosphatase was distributed between two peaks on the gradient. The low-density (1.10–1.15 g·cm^?3) alkaline phosphatase component co-sedimented with the plasma membrane marker, 5′-nucleotidase, had an apparent K_m for 4-methylumbelliferyl phosphate of approx. 59 μM, and was inhibited by levamisole. The high-density (1.20–1.25 g·cm^?3) peak was resistant to levamisole-inhibition, had an apparent K_m of approx. 30 μM and its distribution was distinct from plasma membrane, Golgi, lysosome, endoplasmic reticulum, mitochondria and prolactin granule markers on the isopycnic gradients.     

Hydrangea mosaic virus, a new ilarvirus from Hydrangea macrophylla (Saxifragaceae)

A previously unrecognised virus isolated from Hydrangea macrophylla with chlorotic mosaic leaf symptoms in West Sussex was named hydrangea mosaic virus (HydMV). HydMV was mechanically transmitted without difficulty to four of 16 species from three of five families, and was seed transmitted in Chenopodium quinoa, but was not aphid transmitted. Although relatively unstable in vitro, HydMV was purified by clarifying leaf extracts by emulsification with chloroform and acidification with citric acid, followed by differential centrifugation and sucrose density gradient centrifugation. Purified virus incompletely separated on sucrose density gradients into three components (T. M and B) with sedimentation coefficients (s^o_20w) of 86, 97, and 105 S respectively, but all particles had buoyant densities in caesium chloride of 1.37 g/cm³. Virus contained a single polypeptide species (mol. wt 26.4 times 10³), appeared quasiisometric to ovoid or elliptical, and measured c. 28 times 30 (T), 30 times 30 (M) or 30 times 32–38 nm (B). Single-stranded RNA species or mol. wt 1–25, 1–08, 0–83, 0–36 and 0–27 (RNA-1 to 5 respectively) were obtained from virus preparations but mixtures containing RNA-1 to 3 plus either RNA-4 + 5 or the coat protein, were infective. These properties suggest that HydMV has affinities with ilarviruses, but it showed no serological relationship to any of six ilarviruses or 42 other viruses.     

Separation of hepatocytes from suspensions of mouse liver cells using programmed gradient sedimentation in gradients of ficoll in tissue sulture medium

Liver cells were obtained in suspension using a solution of lysozyme in Joklik's modification of minimum essential medium. Hepatocytes were separated in 74.2 ± 12.9% purity from other liver cells having different densities using isopycnic centrifugation, in 97.1 ± 1.9% purity from other liver cells having different diameters using velocity or rate-zonal centrifugation. A previously reported computer integration of the differential sedimentation equation was employed in determining the gradient design and the speed and duration of centrifugation which would permit purification of hepatocytes from other liver cells. More than 98% of the hepatocytes separated by velocity sedimentation excluded trypan blue. Velocity sedimentation is superior to isopycnic centrifugation for the separation of hepatocytes from liver cell suspensions because it gives more highly purified hepatocytes and because it requires lower centrifugal forces for shorter periods of time.     

Properties of a host range and coat protein variant of broad bean true mosaic comovirus from Vicia faba

Unlike other described isolates of broad bean true mosaic comovirus (BBTMV), a variant, code name SB, infected some non-leguminous plant species and, in N. benthamiana, induced systemic mottling and puckering of the leaves. However, like other described BBTMV isolates, purified SB particle preparations contained isometric particles c. 28 nm in diameter that sedimented as two nucleoprotein components with S_{20, w} values of 90S and 109S; some preparations occasionally contained a component of c. 50S. Virus particles contained two ssRNA species which, when denatured in glyoxal, had estimated M_T values of 2.1 × 10⁶ and 1.3 × 10⁶ and co-electrophoresed with cowpea mosaic virus RNA-1 and RNA-2 respectively. Isolate SB was serologically indistinguishable from British and German isolates of BBTMV. However, SB virus particles contained a major polypeptide (L) of M_r between c. 31 000 and up to three minor ones (S) or M_r between c. 20 000 and 24 000. This contrasts with protein preparations from other BBTMV isolates that typically contain only two polypeptides of M_r c. 37 000 (L) and 21 000 (S). Following isopycnic centrifugation in CsCl, SB particles purified from pea separated into two major components with densities of 1.39 and 1.44 g cm^-3 and a minor component of estimated density 1.43 g cm^-3. In Cs₂SO₄, virus preparations separated into three major components with densities of 1.30, 1.32 and 1.36 g cm^-3 and a minor one of density 1.27 g cm^-3. In CsCl isopycnic gradients, SB particles purified from TV. benthamiana separated into two components with densities of 1.38 and 1.43 g cm^-3. During immuno-electrophoresis in agarose gels, freshly prepared virus and preparations stored for up to 4 days at 4°C contained a single component that migrated rapidly to the anode, whereas similar preparations of an English isolate of BBTMV migrated as a single component that moved only slowly toward the anode but which, within 48 h, contained an additional component with a migration rate similar to that of isolate SB. Isolate SB is therefore a host range variant of BBTMV which, in comparison with previously described isolates of BBTMV, has an increased negative charge of its particles prior to any appreciable degradation of its S protein, and S protein that is degraded less rapidly. These features probably account for the anomalies observed in isopycnic centrifugation.