Low Centrifugal Forces Research Articles

In vitro effect of cholesterol on calcifying activity of vesicles isolated from rabbit aortas

It has been shown that vesicles play a key role in the onset mechanism of aortic calcification related to cholesterol-induced atherosclerosis. This study using a rabbit model was conducted to determine whether cholesterol exerts a direct effect on vesicle's calcifiability. Inclusion of cholesterol in calcifying media stimulated ATP-initiated deposition of calcium in a dose-dependent manner by vesicles isolated from normal aortas using crude collagenase digestion. By contrast, cholesterol did not significantly affect ATP-promoted calcification if vesicles were isolated from atherosclerotic aortas. To determine whether high cholesterol levels in atherosclerotic vesicle preparations may have already maximized calcifying activity and therefore account for lack of the vesicle's response to the sterol, Fourier transform infrared spectroscopy (FT-IR) was used to compare the cholesterol contents in control and atherosclerotic vesicles. The spectral patterns revealed higher levels of cholesterol in vesicle preparations from atherosclerotic aortas than those from normal aortas. Removal of extra-vesicular cholesterol micelles from atherosclerotic vesicles by a relatively low centrifugal force sensitized the vesicles to cholesterol stimulation causing a 2-fold increase in calcifying activity. Of various oxidized forms of cholesterol tested, 7-keto and 6-keto cholesterol enhanced the activity by 2-fold. Altogether, these observations suggest that cholesterol and especially its oxidized forms may induce aortic calcification by directly enhancing the vesicle's ability to calcify.

Glucose 6-Phosphate Produced by Gluconeogenesis and by Glucokinase Is Equally Effective in Activating Hepatic Glycogen Synthase

Glucose 6-phosphate (Glc-6-P) produced in cultured hepatocytes by direct phosphorylation of glucose or by gluconeogenesis from dihydroxyacetone (DHA) was equally effective in activating glycogen synthase (GS). However, glycogen accumulation was higher in hepatocytes incubated with glucose than in those treated with DHA. This difference was attributed to decreased futile cycling through GS and glycogen phosphorylase (GP) in the glucose-treated hepatocytes, owing to the partial inactivation of GP induced by glucose. Our results indicate that the gluconeogenic pathway and the glucokinase-mediated phosphorylation of glucose deliver their common product to the same Glc-6-P pool, which is accessible to liver GS. As observed in the treatment with glucose, incubation of cultured hepatocytes with DHA caused the translocation of GS from a uniform cytoplasmic distribution to the hepatocyte periphery and a similar pattern of glycogen deposition. We hypothesize that Glc-6-P has a major role in glycogen metabolism not only by determining the activation state of GS but also by controlling its subcellular distribution in the hepatocyte.

Liposomal budesonide for dry powder inhaler: Preparation and stabilization

The purpose of the study was to prepare stable liposomally entrapped budesonide (BUD) for a dry powder inhaler (DPI) formulation. BUD liposomes composed of egg phosphatidyl choline and cholesterol were prepared by lipid film hydration technique and sonicated to have the desired size (<5 μm). A rapid method was used for separation of free drug by centrifugation at a lower centrifugal force (G value). Liposomal dispersion was subjected to lyophilization after blending BUD with cryoprotectant in varying bulk and mass ratios, and percent drug remaining entrapped after lyophilization was optimized. Comparative drug retention studies on storage of DPI formulations were carried out in accordance with International Conference on Harmonization guidelines. Critical relative humidity of the formulations was determined and reported as one of the manufacturing controls. Sucrose was found to be the most effective cryoprotectant when present on both sides of the lamellae of liposomes in a bulk strength of 500 mM and mass ratio of lipid:sugar; 1∶10. Blending of sorbolac before lyophilization showed better retention of encapsulated drug (95.59%). The respirable fraction of the product (20.69±1.50%) was comparable with that of the control (26.49±1.52%), suggesting that the liposomal BUD can be successfully delivered throughout the broncho-pulmonary tree. The findings demonstrate that liposome of BUD can be prepared with a high entrapment value, stabilized by lyophilization, and delivered as an aerosolized DPI. The stability studies of lyophilized product suggests a shelf-life of one year when stored under refrigeration (2°C–8°C).

Liposomal budesonide for dry powder inhaler: preparation and stabilization.

The purpose of the study was to prepare stable liposomally entrapped budesonide (BUD) for a dry powder inhaler (DPI) formulation. BUD liposomes composed of egg phosphatidyl choline and cholesterol were prepared by lipid film hydration technique and sonicated to have the desired size (< 5 micro m). A rapid method was used for separation of free drug by centrifugation at a lower centrifugal force (G value). Liposomal dispersion was subjected to lyophilization after blending BUD with cryoprotectant in varying bulk and mass ratios, and percent drug remaining entrapped after lyophilization was optimized. Comparative drug retention studies on storage of DPI formulations were carried out in accordance with International Conference on Harmonization guidelines. Critical relative humidity of the formulations was determined and reported as one of the manufacturing controls. Sucrose was found to be the most effective cryoprotectant when present on both sides of the lamellae of liposomes in a bulk strength of 500 mM and mass ratio of lipid:sugar; 1:10. Blending of sorbolac before lyophilization showed better retention of encapsulated drug (95.59%). The respirable fraction of the product (20.69 +/- 1.50%) was comparable with that of the control (26.49 +/- 1.52%), suggesting that the liposomal BUD can be successfully delivered throughout the broncho-pulmonary tree. The findings demonstrate that liposome of BUD can be prepared with a high entrapment value, stabilized by lyophilization, and delivered as an aerosolized DPI. The stability studies of lyophilized product suggests a shelf-life of one year when stored under refrigeration (2 degrees C-8 degrees C).

Selective retension of active cells employing low centrifugal force at the medium change during suspension culture of Chinese hamster ovary cells producing tPA

The effect of centrifugal force applied for cell separation at the medium change on the growth, metabolism and tissue plasminogen activator (tPA) productivity of Chinese hamster ovary (CHO) cells suspension culture was investigated. The viability of the precipitated cells increased exponentially as the centrifugal force decreased. However, the cell recovery was lower than 91% when centrifugal forces applied for 5 min was less than 67 × g. In cultures incubated for 474 h with 7 medium changes employing centrifugal forces ranging from 67 to 364 × g, a centrifugal force lower than 119 × g resulted in higher specific rates of growth, glucose consumption, and lactate and tPA production during the whole culture period. On the other hand, daily centrifugation at 67 to 537 × g without discarding the supernatant had no effect on the specific rates. The cultures inoculated with cells precipitated at a centrifugal force of 67 × g showed apparently higher specific rates of metabolism compared to those inoculated with cells in the supernatant. The cells in the supernatant and the precipitate obtained following centrifugation at 67 × g have average diameters of 15.5 and 17.4 μm, respectively. The intracellular contents of amino acids, especially nonessential amino acids, of the precipitated cells were markedly higher than those of the cells in the supernatant. These results indicate that large cells with high amino acid content and metabolic activity were selectively retained in the culture by means of centrifugation at low forces such as 67 × g. Consequently, application of a low centrifugal force is recommended for medium change in order to maintain higher specific productivity of suspended mammalian cells in perfusion culture.

Coprecipitating ammonium–iron(iii)–hexacyanoferrate(ii) from aqueous dispersion with albumin and trichloroacetic acid

Ammonium–iron(III)–hexacyanoferrate( II) (AFCF) is a caesium-binding agent used, for example, to prevent the absorption of radioactive caesium from the alimentary tract of domestic animals. Owing to its colloidal nature, its separation from aqueous solution with standard methods is tedious, complicating in vitro studies. A new and simple method for separating AFCF from water and water-soluble components was developed, based on quantitative coprecipitation with bovine albumin and trichloroacetic acid (TCA). The resulting precipitate and supernatant can be separated with low centrifugal forces. Bound caesium follows AFCF into the precipitate, whereas free caesium remains in solution. This makes the method a potential tool in the study of the thermodynamic and kinetic properties of AFCF–caesium interactions in vitro. Effects of different factors, such as concentrations of components, speed and duration of centrifugation and temperature, are described.

DESTABILIZATION OF WATER IN BITUMEN EMULSION BY WASHING WITH WATER

ABSTRACT In order to get more information about the mechanism of stabilization of water-in-diluted-bitumen emulsion, bitumen diluted with toluene (10%, 25%, and 50% in volume) was “washed” using different amounts of water (0.20% in volume). The washing water was emulsified and then separated by high-speed ultra-centrifugation. The supernatant was then used to create a second w/o emulsion with the addition of new water. Stability of the new emulsion was measured in terms of the water separation rate under a low centrifugal force It has been found that a very stable w/o emulsion was obtained in original diluted bitumen. However, after the diluted bitumen was pre-washed with a few per cent of water, the second emulsion became unstable. This indicates a significant effect of pre-washing with water on emulsion stability, possibly through the removal of emulsion stabilizing agents. Based on analytical results such as surface tension, and FTIR spectra, it appears that a small fraction of bitumen, mostly polar compounds such as carbonic acids and other oxygen-containing compounds, are responsible for the emulsion stabilization. Emulsion stability decreases with increasing volume of washing water but increases with the bitumen concentration.

The influence of triploidy on gene expression in the silkworm, bombyx mori

In Bombyx mori, it is well established that polyploids are easily induced when newly laid eggs are exposed to a variety of conditions, such as high or low temperature, centrifugal force, or chemicals like colchicine. To investigate gene dosage effects by varying the ploidy, the transcription levels of six genes expressed in various tissues were analysed in the diploid and two different genetically produced triploids (PPC and CCP). In the PPC triploid, the transcription level per cell of two genes was directly proportional to the structural gene dosage, whereas two other genes showed the mRNA level expected if compensation occurred. In the CCP triploid, three genes displayed dose-dependent levels of expression, whereas one gene showed the same expression level as the diploid strains. In both triploids, exceptional cases showed a negative correlation of expression with ploidy or a positive correlation greater than expected from the structural gene dosage. Interestingly, the transcription levels of most tested genes were significantly different from the strains which were used as parents of the triploids, and also widely divergent expression patterns were found for some genes in the diploid offspring. In this study, the cause of the unexpected expression patterns observed in the euploid series is discussed in relation to the difference between the two parental strains in expression level of genes and in trans-acting regulatory effects on their target genes.

A Dilatancy Assay for Nucleoid Denaturation: The Centrifugation-Dependent Clumping of Denatured Spermidine Nucleoids

The ability of low centrifugal forces to convert denatured spermidine nucleoids fromEscherichia coliinto nonsedimentable macroscopic clumps is the basis of a rapid and simple, nonisotopic assay for nucleoid denaturation.

A New Experimental Method to Study Combined Fatigue of Actual Turbine Disk Mortise Teeth at Elevated Temperatures

This paper presents a new experimental system to study the L-HCCF of an actual turbine disk mortise teeth at elevated temperature, using an actual disk as experimental component. This system ingeniously achieves combined loading (simulating low cycle radial centrifugal force and high cycle crosswise vibration of blade), high-frequency induction local heating (550°C constant temperature), control of high cycle vibrating frequency and amplitude, and crack real-time detection. The experimental result is identical with the practical flight failure. This method can be easily popularized to study the L-HCCF of many components.

Rapid determination by centrifugal ultrafiltration of inter-mixed micellar/vesicular (non-lecithin-associated) bile salt concentrations in model bile: influence of Donnan equilibrium effects

We have developed the technique of rapid (< 2 h) centrifugal ultrafiltration to measure the inter-mixed micellar/vesicular (non-lecithin-associated) bile salt (BS) concentrations (IMC) of individual BS in model biles. This methodology uses a centrifugal concentrator with a reinforced membrane, through which a small fraction (< 15% total volume) of model biles was ultrafiltered by low centrifugal forces (1500 g 5-60 min). Total and individual BS concentrations in the filtrate were measured by high performance liquid chromatography. However, non-filterable anions, in this case BS/lecithin/cholesterol mixed micelles and unilamellar vesicles, induce an asymmetric distribution of ions across the membrane. Therefore, BS concentrations in the filtrate exceeded the true IMC, which was estimated taking into account Donnan equilibrium effects. To confirm the hypothesis that a correction for Donnan forces was necessary, distributions of BS and another filterable anion, chloride, were measured in systems containing the non-filterable polyanion dextran sulfate as an analogue for non-filterable polyanionic mixed micelles and vesicles. An asymmetric distribution of the monovalent anions chloride and BS monomers as well as polyvalent simple BS micelles was indeed present during centrifugal ultrafiltration. This new methodology was validated by comparing IMC values with those obtained by modified equilibrium dialysis also corrected for Donnan equilibrium effects (Donovan, J.M., et al. 1991. J. Lipid Res. 32: 1501-1512). Centrifugal ultrafiltration, which utilizes < 1 ml of bile, determines the composition in the IMC necessary to separate micelles and vesicles of native biles by techniques that involve dilution of bile such as gel filtration chromatography.

Evacuolation and enucleation of mesophyll protoplasts in self‐generating percoll gradients

ABSTRACTMesophyll protoplasts from Brassica oleracea, B. napus, Nicotiana tobaccum and Solanum tuberosum were isolated and subjected to uttracentrifugation at 65000g for 30 min in percoll solutions containing various strengths of salt and osmotic stabilizing agents. After centrifugation, the self‐generated percoll gradients were evaluated for their effectiveness in protoplast evacuolation and enucleation. The vacuoles, cell debris, evacuolated protoplasts and enucleated protoplasts were separated. Factors that affected evacuolation and enucleation in the percoll gradients were described. Mesophyll protoplasts produced by epidermis peeling and short enzyme incubation periods were more easily evacuolated and enucleated than those produced by leaf‐slicing and long incubation periods. Lower centrifugal force at 25000g for 80 min was also successful in evacuolating and enucleating the mesophyll protoplasts. A green band that contained nearly pure evacuolated protoplasts, of which 45% were enucleated protoplasts, was obtained from the self‐generated percoll gradient. Rhodamine 123 staining of mitochondria indicated that the evacuolated protoplasts were metabolically active and were capable of regenerating the vacuole and cell wall. Cell divisions were also observed when the evacuolated protoplasts were cultured.

Purification and characterization of annexin proteins from bovine lung

Calcium-dependent association with a detergent-extracted particulate fraction was used as the first step in the purification of a group of phospholipid binding proteins. Elution of the detergent-insoluble fraction with excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) resulted in the release of several soluble proteins, termed calcium-activated proteins or CAPs. In the present paper, we describe the simultaneous purification of these CAPs and characterize their interaction with phospholipid, actin, and calmodulin. Partial sequence analysis has identified the majority of the CAPs as members of the annexin family of calcium and phospholipid binding proteins. Two additional CAPs may be novel proteins, one of which appears to be an annexin protein. All CAPs demonstrated Ca2(+)-dependent binding to phosphatidylserine vesicles but did not bind to phosphatidylcholine vesicles. The majority of CAPs exhibited Ca2(+)-dependent binding to F-actin; however, only CAP-III affected the rate of conversion of G-actin to F-actin. The interaction of CAP-III and lipocortin-85 with F-actin resulted in a Ca2(+)-dependent increase in both light scattering and sedimentation of F-actin under comparatively low centrifugal force. In contrast, only lipocortin-85 caused the formation of F-actin bundles. Although all of the CAPs bound to a calmodulin affinity column in a Ca2(+)-dependent manner, attempts to demonstrate binding of CAPs to native calmodulin were unsuccessful. These studies therefore document the similar behavior of the CAPs toward phospholipid and calmodulin but clearly show that F-actin binding or bundling is not a general property of these proteins. The reported purification procedure should allow further comparative studies of these proteins.

High-strength partially stabilized zirconia compact obtained by centrifugal consolidation

It has been reported that agglomerates present in ceramics powder limit the final density of the sintered compact [1, 2]. This is shown [3] to be due to different sintering kinetics between the inside and outside of an agglomerate and because the interagglomerate pores do not easily disappear even at the final stage of sintering. A technique of powder dispersion in liquid has been investigated [4] as a method for obtaining dense packing of powder particles. Rhodes [2] reported that a highly dense compact is obtained when agglomerates are eliminated by means of ultrasonic dispersion of powder followed by centrifuge cast. In the present study, the starting powder containing agglomerates is dispersed in aqueous solution by means of a highpower ultrasonic vibrator; then a centrifugal force is applied to the slurry, not only for the forced sedimentation, but for the effective compaction of powder. This is aimed at obtaining highly dense bulkcompacts to carry out the measurement of their flexural strength. PSZ powder used (Toyo Soda Co.) is co-precipitated ZRO:2(2.5) mol % Y203 (called 2Y and 2.5Y) having particle size of about 30 to 50 nm. The powder contains agglomerates of several to several tens of micrometres diameter. Firstly, the as-received powder was gently ground with an agate mortar for 15min. Then the powder dispersion was carried out in powder-to-solution ratio of 15g/15ml. For promoting dispersion, ultrasonic vibration (at 1 kW output) was applied to the slurry for 1⁄2h. Besides methanol as an effective organic dispersing solvent [5], Pierre [6] rep~)rted that the lowest viscosity is attained at pH = 1.2. In the present study, a sedimentation experiment was carried out for aqueous-HNO3 solution with different pH; the result suggests that solution at pH = 3 suspends the powder well with the least amount of sediments, as shown in Fig. 1. (Dispersion at pH = 11, 7, 3, 1.5 was tried and the porosities of the sintered compacts examined. The lowest porosity was obtained in the case of pH = 3.) Because undispersed agglomerates still remain after dispersion treatment, a sedimentation-classifying procedure was performed for reasons given below. The slurry was diluted 10 times and, after stirring, it was sedimented and the suspension was decanted. The lower centrifugal force (< 10000G) was applied for 10min for collection of the powder and the higher (20 000 G) force was applied for 30 min to the condensed slurry for final compaction. Much higher centrifugal force (200 000G) has been shown to result in

A procedure for extracting staphylococcal enterotoxin from foods at elevated temperatures and low centrifugal forces

Various low relative centrifugal forces (RCFs) were tested for their effectiveness in ‘cleaning up’ extracts of staphylococcal enterotoxin from food homogenates. RCFs of at least 10 000 × g were effective if centrifugations were for 30 min periods or longer. Below 8000 × g, and down to about 1000 × g, the cleaning up of extracts was effective only if centrifugation was preceded by filtration of food homogenates. Investigations of the thermal stability of enterotoxin during cleaning up of extract at higher than refrigeration temperatures showed that centrifugation temperatures of up to 40°C had little adverse effect on the serological activity of the toxin. Based on these findings a procedure was devised for extracting enterotoxin from foods using basic bench top centrifuge at ambient temperatures. Separation of enterotoxin from food proteins in the extract was achieved by carboxymethyl cellulose-column chromatography. Levels of enterotoxin A detectable by the microslide gel double diffusion method following extraction by this procedure were 2.5 ng/ml for milk, 2.5 ng/g for yogurt and 5 ng/g for cheese and corned beef.

?-1-4-and ?-1-3-glucan synthases are associated with the plasma membrane of the fungus Saprolegnia

Cytoplasmic membranes from mycelium or protoplasts of Saprolegnia monoica (a cellulosic cell-wall fungus) were separated by continuous sucrose-density-gradient centrifugation. Glucan synthases assayed at low (micromolar uridine 5'-diphosphate (UDP) glucose for β-1-4-glucan synthase) and high (millimolar UDP glucose for β-1-3-glucan synthase) substrate concentrations were associated with membranes exhibiting vanadate-sensitive, oligomycin-insensitive ATPase and equilibrating at density 1.16 g cm(-3). Synthase activities were also bound to membranes of lower density (1.10 and 1.145 g cm(-3)). Plasma membranes were stabilized by coating protoplasts with concanavalin A. After lysis of the protoplasts, plasma membranes recovered by low centrifugal forces were isolated in continuous isopycinic gradients. Both synthase activities peaked with [(3)H]concanavalin A and Na-vanadate ATPase indicating that the synthetases are located at the plasma membrane. Treatments of intact protoplasts with cold glutaraldehyde or proteases before disruption lead to a diminution of glucan-synthase activities indicating that at least part of the enzymes of plasma membrane face the outside of the cell.

Isolation of plasma membrane from protoplasts of Lolium multiflorum (ryegrass) endosperm cells.

Plasma membranes have been isolated from protoplasts of suspension-cultured ryegrass (Lolium multiflorum) endosperm cells. The protoplast membrane is coated before cell disruption with murine myeloma protein J539, a galactose-binding immunoglobulin A. The plasma membrane is labelled with 125I by using chemically or enzymically catalysed iodination techniques, or, more conveniently, by using 125I-labelled myeloma protein J539, which enables the membrane to be simultaneously coated and labelled. Protoplast lysis is effected by gentle mechanical means after swelling in hypo-osmotic medium. The plasma-membrane fraction is recovered at low centrifugal forces by fractionation of cell lysates on a discontinuous sucrose/sorbitol gradient. The plasma-membrane fraction is enriched 96-fold on a protein basis with respect to the specific radioactivity of 125I-labeled myeloma protein J539 in the homogenate. Electron microscopy showed long membrane profiles often associated with one another.

Evidence for two particulate cholesterol ester hydrolase enzymes in rat corpus luteum

Two cholesterol ester hydrolases (CEH) were detected in the particulate fraction of the rat corpus luteum cell, one sedimenting at low centrifugal forces (between 760 and 25,410 g) and the other at higher centrifugal forces (25,410–124,000 g). The CEH in the fractions sedimenting at low centrifugal forces had an activity of 20–70 pmol/min/mg protein. This enzyme showed a similar sedimentation profile to the lysosomal acid phosphatase enzyme. A second, more active (200–350 pmol/min/mg protein) CEH activity had a different pH optimum and sedimented over higher centrifugal forces. These findings suggest that two CEH can provide cholesterol for steroidogenesis, using as substrate either cholesterol esters stored in the cell, or cholesterol esters derived from plasma lipoprotein.

Isolation and characterization of a calcium-sensitive alpha-actinin-like protein from human platelet cytoskeletons.

Platelet cytoskeletons were isolated by extracting these highly contractile cells with a solution containing 1% Triton X-100 and 10 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid as recently described (Rosenberg, S., Stracher, A., and Lucas, R. C. (1981) J. Cell Biol. 91, 201-211). The Triton-insoluble cytoskeleton consists mostly of actin, a high molecular weight actin-binding protein and a previously unidentified protein with an apparent molecular weight on sodium dodecyl sulfate gels of 105,000 (+/- 5,000). We describe the purification of this 105,000-dalton protein from the platelet cytoskeleton using ammonium sulfate fractionation and ion exchange chromatography. This 105,000-dalton protein was found to cross-react with antibodies to beef cardiac alpha-actinin. One-dimensional partial proteolysis maps showed similarity to, but not identity with, the major peptides of the platelet 105,000-dalton protein and skeletal muscle alpha-actinin. The platelet 105,000-dalton cytoskeletal protein binds to and causes the sedimentation of skeletal muscle F-actin under comparatively low centrifugal force. This process, however, is inhibited by calcium ions, unlike the binding of any of the muscle alpha-actinins described to date. Thus, it is likely that the 105,000-dalton protein is the platelet form of alpha-actinin, its different structure accounting for its different actin-binding behavior.

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.