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Density gradients prepared from colloidal silica particles coated by polyvinylpyrrolidone (Percoll)
ANALYTICAL
BIOCHEMISTRY
88,271-282
(1978)
Density Gradients Prepared from Colloidal Silica Particles Coated by Polyvinylpyrrolidone (Percoll) HWKAN
PERTOFT,
TORVARD C. LAURENT, LENNART KAGEDAL
Institute ofMedical and Physiological Box 575, S-751 23 and Pharmacia
Chemistry, Universiry Fine Chemicals, Box
TORGNY
LAAs AND
of Uppsala, Biomedical 175 S-751 04 Uppsala,
Center, Sweden
Received May 23. 1977; accepted February 10, 1978 A new gradient medium (Percoll) for density gradient centrifugation of cells and subcellular particles is described. It consists of colloidal silica particles which have been firmly coated with a layer of polyvinylpyrrolidone. The particle population is polydisperse, and the average diameter is approximately 17 nm. Both the electrophoretic mobility of the particles and the conductivity of the solution are low, indicating a low surface charge. The colloid has a high solubility and forms clear solutions. It can be used in concentrations which give solution densities between 1.00 and 1.20 g/ml. The solutions have a low osmolality and can be mixed with electrolytes of physiological pH and ionic strength. The sedimentation properties of the particles are described. Density gradients can be formed by highspeed centrifugation. The colloid has been shown to be nontoxic for a number of cells and cell organelles.
Separation of cells and cell organelles by isopycnic gradient centrifugation is a potentially powerful technique, the application of which has been somewhat limited by the lack of suitable gradient materials. Most available substances form solutions of undesirably high osmolality or viscosity. Silica sols were first used by Mateyko and Kopac (1) for the “isopycnic cushioning” of cells from animals. Subsequently, Pertoft and co-workers [see Pertoft and Laurent (2)] established the general applicability of silica sols in density gradient centrifugation in a systematic investigation of the conditions for the separation of cells, viruses, and a variety of other particles. The applications have been somewhat limited, however, by the toxic effects of the silica. Pertoft (3) and Wolff and Pertoft (4) have described methods to prevent these effects, by adding polymers which are adsorbed on the surface of the silica particles. The presence of polymer in the solutions does, however, increase its viscosity, and it is also difficult to remove the polymer from the biological material after centrifugation. In this paper we describe the characteristics and uses of silica particles coated with a layer of polyvinylpyrrolidone (PVP). This product (Percoll) has many properties which renders it suitable for use in isopycnic separa271
0003-2697/78/0881-0271$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
272
PERTOFT
ET AL.
tion of cells and subcellular material. Water solutions of the product are of high density, low osmolality, and low viscosity. Generation of gradients is an automatic process occurring during centrifugation of the colloid. MATERIALS
AND METHODS
Colloidal silica ~01s. Pure colloidal silica, Ludox HS, was obtained from Du Pont de Nemours (Wilmington, Delaware). It was dialyzed repeatedly against distilled water before use. Colloidal silica coated with polyvinylpyrrohdone (Percoll) is a product of Pharmacia Fine Chemicals (Uppsala, Sweden). PEL-medium. PEL-medium was made according to Wolff and Pertoft (4) by mixing Ludox HS [12% (w/v)], polyvinylpyrrolidone (PVP) [5.3% (w/v); MW 40,000; from A. H. Thomas, Philadelphia, Pennsylvania], and Eagle’s minimum essential medium. Polyethylene glycol. Polyethylene glycol (PEG) (MW, 4000) was purchased from AB KEBO (Stockholm, Sweden). Chemical methods. Dry weight analysis of the particles was performed (heating to 100°C over phosphorus pentoxide in vacua to constant weight), and PVP content was calculated from nitrogen determinations according to Kjeldahl . Gel chromatography. Gel chromatography was performed at 4°C on Sepharose 4B using laboratory columns type K 15/90 from Pharmacia Fine Chemicals AB. A Uvicord III (LKB, Stockholm) was used to record the chromatograms by measuring transmission at 206 and 225 nm. Analytical ultracentrifugation. A Spinco Model E Analytical Ultracentrifuge equipped with schlieren optics was used to analyze the sedimentation behavior of silica particles. The experiments were performed in 12-mm double sector cells at a rotor speed of 12,000 rpm and a temperature of 20°C. Preparative ultracentrifugation. The following centrifuges were used to generate density gradients in Percoll: Beckman ultracentrifuges L4,L2-65B, and L50 equipped with the angle rotors 35, 40.2, and 65 and the swingout rotor 36; an MSE Super Speed 75 equipped with an 8 x 14 ml titanium angle rotor, (MSE), a 10 x loo-ml aluminum angle rotor (MSE), and a 6 x 38-ml aluminum swing-out rotor (MSE); an MSE 25 centrifuge with a 6 x 100 ml angle rotor; a Sorvall RC2-B centrifuge with an SS 34 angle rotor. Some experiments were performed in which sucrose to a final concentration of 0.25 M or sodium chloride to a final concentration of 0.15 M were added to the colloid. The samples were run for 10 to 60 min at speeds between 10,000 and 50,000 rpm and at a temperature of 20°C. The tubes were then fractionated by pumping Ludox HS to the bottom of the tube, and fractions obtained from the top were analyzed for density.
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273
Electron microscopy. A negative contrast technique was used, with 1% uranyl acetate in 0.2 M ammonium acetate (pH 4.6) as a contrasting agent. The specimens were examined in a Siemens Elmiskop I at a magnification of 80,000 using an operating voltage of 60 kV. A Zeiss particle analyzer was used to measure the distribution of particle diameters from the micrographs. Free zone electrophoresis. Free zone electrophoresis was carried out, as described by Hjerten (5). The runs were performed in 0.15 M sodium acetate (PH 4), 0.01 M phosphate buffer (pH 7.4), and 0.13 M NaCl, or 0.15 M Tris buffer (pH 9.0). The current was kept between 5 and 7 mA, and the voltage was between 300 and 1400 V. The glass tube was scanned every Iifth minute, and the absorbance at 280 nm and 320 nm was recorded. The migration of the samples was followed during 1 hr. Osmolality measurements. Osmolality measurements were performed in a freezing-point osmometer from Advanced Instrument Inc, Newton Highlands, Massachusetts). Conductivity. Conductivity was measured in a Conductivity Meter CDM 3 from Radiometer (Copenhagen, Denmark). Viscosity. Viscosity relative to water was measured in a KPG Ubbelohde Viscometer (JenaER Glas, Schott & Mainz, West Germany) at 20°C. Density. Density was measured in organic density columns (6) or in a densitometer (DMA 10 Digital Densimeter, Anton Paar, Graz, Austria). Refractive index. The refractive index was mesured in an Abbe refractometer. RESULTS
Chemical analysis of Percoll. Percoll is composed of solid silica spheres coated with PVP. The nitrogen content of the freeze-dried material is close to 1.50%, corresponding to 11.9% PVP. The PVP content varied between 11.4% and 12.4% in six different preparations. Gel chromatography. Gel chromatography was used to make a comparison between Percoll and a mixture of colloidal silica, PVP, and Eagle’s medium (PEL-medium (4)]. PVP was measured by its uv-absorption, and the silica was identified by density measurements on the fractions. In the Percoll preparation the PVP and silica material overlapped, indicating that PVP was firmly bound to the silica (Fig. 1). There was a small peak (amounting to between 1.2 and 3.2% as measured in different batches) close to the total volume, which was considered to be free PVP. When the main peak was rechromatographed, no material appeared at Vt. The PEL-medium showed a very different pattern (Fig. 1). A large uv-absorbing peak at the end of the chromatogram indicated that free PVP was present. There was some PVP bound to the silica, but the material showed a great heterogeneity.
274
PERTOFT ET AL. PEL
cq
“I
1
, PERCOLL
0
100
59
v, EFFLUEN
1
VOLUME
t vt (ml)
‘,
150
FIG. 1. Top: Gel chromatography of 2 ml of a mixture containing 12% (w/v) Ludox HS, 5.3% (w/v) PVP, and Eagle’s MEM [PEL-medium (4)] on a 1.5 x 85cm column of Sepharose 4B. The flow rate was 0.2 mUmin, and the eluent was 0.13 M NaCl + 0.01 M phosphate buffer (pH 7.4). PVP was analyzed by absorbance, and silica was analyzed by density measurements. Bottom: Gel chromatography of Percoll under the same experimental conditions.
It is known that polyethylene glycol can desorb polyesters which are adsorbed to silica surfaces (7). Therefore an experiment similar to that shown in Fig. 1 was carried out on Percoll with 2% PEG present in the eluant. This revealed no increase in the size of the small peak at the total volume, indicating that PEG had not been able to desorb PVP from silica. TABLE PROPERTIES
OF PERCOLL
AND DIALYZED
LUDOX
1 HS OF EQUAL
DENSITIES
(1.130
g/ml)
Colloidal solutions Property
Percoll”
Ludox HSb
Silica content (%, w/w) PVP content (%, w/w) Refractive index PH Viscosity (cP) Osmolality (mosM/kg of H,O) Conductivity (mS/cm)
20.0 2.7 1.3540 + 0.0005 8.8 k 0.1 9- 14 10 5 2 0.60 + 0.02
20.0 1.3485 + 0.0005 10 ” 0.2 2 + 0.2 20 + 2 1.50 k 0.02
U Figures are based on measurements of six batches of density, 1.130 + 0.005. * Figures are based on three batches of dialyzed Ludox HS. The values are not the same as those for undialyzed Ludox HS [compare Pertoft and Laurent, Ref. (9)].
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MATERIAL
Properties of Percoll solutions. Concentrated solutions of Percoll (up to a density of 1.2 g/ml) are colorless, slightly opaque liquids. They can be autoclaved (12O”C, 20 min) without any change in the properties. Table 1 shows a comparison between various properties of Percoll and those of a preparation of the original silica sol carefully dialyzed against distilled water. Although the PVP coat has no visible effect on the density of the solution, it can be seen that it does cause a
FIG. acetate
2. Electron at pH 4.6.
microscopy
of Percoll
particles
negatively
contrasted
with
1% uranyl
276
PERTOPT ET AL.
lowering of the osmolality and of the conductivity of the particles. This is supposedly due to a decrease in surface charge, which is also observed in electrophoresis. The relative viscosity of the solutions increases as a result of the polymer coating, but is still low for a density gradient medium. Electron microscopy. Percoll particles are shown in Fig. 2. They are not as aggregated, as is often found with pure silica particles [compare Fig. 1, Pertoft and Laurent (2)]. The colloid is polydisperse, as shown in Fig. 3. The mean particle diameter was determined to be 17.2 nm. The standard deviation of the size distribution was 2.3 nm. An identical result was obtained for a Ludox HS batch. Analytical ultracentrifugation. Both the Percoll and the Ludox particles sedimented as single peaks in the ultracentrifuge (Fig. 4). The concentration dependence of the sedimentation coefficient of Percoll is different from that of the uncoated particles (Fig. 5), there being a more rapid drop in the s02D,Wat low concentrations for coated than for uncoated particles. The sedimentation is also dependent on the ionic strength of the medium (see below). Electrophoresis. In free zone electrophoresis carried out at physiological ionic strength and pH 7.4, the mobility of the Percoll particles is only one tenth that of the original silica sol (Fig. 6A). The mobility of both Perco11 and Ludox HS varies with pH, and an isoelectric point of about 3 is indicated. This is in agreement with published data for colloidal silica (8). Variation of physical-chemical parameters with ionic strength. The viscosity of Percoll and of Ludox HS solutions, measured in water and in 0.15 M NaCl, is shown in Fig. 7. It is seen that the viscosity of
Diameter
(i-m)
FIG. 3. Percoil (-) and Ludox HS (-----) particles were examined in the electron microscope (magnification 68,000X), and by utilizing a particle analyzer on the photographic plates, the number of particles and the particle diameters were measured. The diameter was 17.3 nm for Percoll and 17.2 nm for Ludox HS. The standard deviation was 2.3 nm in both cases.
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MATERIAL
A
FIG. 4. Analytical ultracentrifugation of coated and uncoated colloidal silica at a rotor speed of 12,000 rpm and a concentration of 3%. The wedge cell (upper curve) has 0.15 M NaCl as solvent and the standard cell (bottom curve) has water as solvent. (A) Ludox HS; and (B) Percoll. Pictures were taken with a bar angle of 60” after 16 min at full speed.
Percoll solutions is strongly dependent on ionic strength, in contrast to that of Ludox HS. The sedimentation coefficient of Percoll was measured in sodium chloride of various ionic strengths (Fig. 5). In both dilute and concentrated solutions of Percoll, the particles sediment slower at low PERCOLL
I 0
LUDOX-HS
I
70 CONCENTRATION
20
0 OF
10 COLLOIO
M I%.
w/wl
FIG. 5. The sedimentation constant of Percoll and of Ludox HS as a function of concentration. The runs were made in 0.15 M NaCl (0 0); 0.01 M NaCl (0 0); 0.001 M NaCl (m n ); and distilled water (0 0).
278
PERTOFT
T/ME
ET AL.
fsec
. IO-?,
A
PH
FIG. 6. (A) Free zone electrophoresis of Percoll (0 0) and Ludox HS (0 0). A 10-~1 sample of a particle suspension with a density of 1.023 g/ml was applied in each run. The experiments were carried out in 0.01 M phosphate buffer (pH 7.4) + 0.13 M NaCI. Voltage, 300 V; current, 6 mA; and temperature of the cooling water, 10°C. Scans were made regularly up to 4200 set, and the migration distance versus time is shown. (B) The mobility of particles of Ludox HS and of three different batches of Percoll was measured in several runs at pH 4 in 0.15 M sodium acetate buffer; at pH 7.4 in 0.01 M phosphate buffer + 0.13 M NaCI; and at pH 9 in 0.15 M Tris buffer. Experimental details are described in (A).
ionic strength than they do at physiological salt concentration. This phenomenon is more pronounced for Percoll than for Ludox HS. Gel chromatography performed as described in the legend to Fig. 1 but with distilled water as eluent resulted in a displacement of the Percoll peak toward the void volume. Pure Ludox HS cannot be chromatographed, as it adsorbs to the Sepharose. The results from viscosity measurements, gel chromatography, and sedimentation studies indicate that the Percoll particles occupy a larger hydrodynamic volume when the ionic strength is decreased. Free zone electrophoresis was performed in 0.02 M phosphate buffer (pH 7.4) and in the same buffer with the addition of 0.03, 0.13, or 0.28 M NaCl. There was an increase in mobility with
A NEW DENSITY
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OF
COLLOID
MATERIAL
279
(%.‘+‘/w)
FIG. 7. The viscosity of Percoll and Ludox HS solutions as a function of concentration. The measurements were made in distilled water (0 0) and in 0.15 M NaCl(0 __ 0).
decreasing ionic strength (from 1 x 10-j to 5 x 1O-5 cm2.sec-’ *V-l), but the experiments at low salt concentration were technically difficult to perform. It was not possible to compare the effect of ionic strength on Percoll with that on pure colloidal silica at ionic strengths above 0.2 M because of the instability of the latter (2). Generation of density gradients of Percoll. Formation of density gradients of Percoll can be performed by conventional methods, but it can also be done by high-speed centrifugation. The gradients are the result of sedimentation of the colloidal particles. Because the material is polydisperse (Figs. 2 and 3), the individual particles sediment with different speeds, resulting in a smooth concentration gradient. Examples of gradients generated in the centrifuge are shown in Fig. 8A. These examples also demonstrate that the slope of the gradients which are formed is essentially independent of the starting density of Percoll. It could also be shown empirically that the slope of the gradient generated is approximately linearly related to the time and g-force of the centrifugation (Fig. 8B). Other factors which influence the shape of the gradients are the size of the centrifuge tubes, the geometry of the rotor, and the solvent in which Percoll is dissolved. Table 2 summarizes a large number of experiments in which gradients were generated under various experimental conditions, and it may serve as a guide in selection of centrifugation conditions. DISCUSSION
The present work is based on the earlier observation that colloidal silica solutions can be used for density gradient centrifugations and that the toxic effects of the silica can be inhibited by adding polymers such
280
PERTOFT ET AL.
M DISTANCE
A
B
FROM
g-FORCE
THE
MENISUS
I MIN
B (cm)
I lo5
FIG. 8. Development of density gradients during centrifugation of Percoll solutions. (A) Different concentrations of Percoll dissolved in 0.25 M (isotonic) sucrose were centrifuged in an MSE 8 x 14 ml rotor at an average g-value of 60,000 for 30 min (A A) and 60 min (0 - - - 0). The density gradients formed are shown. The densities of the solutions before the runs are given on the right-hand side. The slopes of the gradients were recorded in the middle of the tubes [see Table 2 and (B)]. M and B denote the menisci and bottoms of the centrifuge tubes. (B) The slope of the density gradient in the middle of centrifuge tubes was recorded after centrifugation of Percoll in 0.25 M sucrose for various times at varying centrifugal forces in two different rotors. The starting density of the Percoll solution varied between 1.05 and 1.16 g/ml, but the gradients which developed show little dependence on the starting density [see (A)]. The slope of the gradient increases approximately linearly with the amount of centrifugation (g-force x time). The runs were made in an MSE rotor (14-ml tubes, angle 29”) (---) and Beckman rotor 6.5 (135ml tubes, angle 23.5”) (-).
as polyethylene glycol, dextran, and polyvinylpyrrolidone (3,4). The polymers were adsorbed to the silica in the presence of an excess of free polymer [see, e.g., Ref. (2)]. The presence of excess polymer in the solution has, however, many drawbacks.
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281
MATERIAL
Percoll is essentially free of unbound PVP, as judged from the gel chromatograms. The PVP coat seems to be firmly attached to the silica surface and cannot be desorbed by polyethylene glycol. The thickness of the PVP coat can be calculated from the following data: The PVP content is analyzed to be 11.9%, the densities of silica and of PVP are 2.2 and 1.2 g/ml, respectively, and the diameter of the silica particles is 17.2 nm. The value of 0.6 to 0.7 nm so calculated is close to monomolecular. No difference in the diameter of coated and uncoated silica could be detected using the electron microscope, but it is possible that the contrasting agent penetrates the coat. If the PVP coat contains water, it would be thicker than 0.7 nm. The coating of the silica gives it many advantages over the pure silica sol. The colloid is more stable, especially in physiological salt solutions; it has a low osmolality, apparently because of a decreased or masked surface charge; it has a low toxicity toward cells; and it can be used to fractionate cell organelles such as lysosomes and viruses (9). Despite a low surface charge, as judged from electrophoresis, the viscosity and sedimentation of Percoll are more dependent on ionic strength than they are for pure silica (Figs. 5 and 7). The apparently larger hydrodynamic volume of the TABLE CHARACTERIZATION
Type of rotor Beckman rotor 6.5 Beckman rotor 65 Beckman rotor 35 Beckman rotor 40.2 Beckman rotor 40.2 Beckman rotor SW 36 MSE rotor 10 x 100 MSE rotor 8 x 14 MSE rotor 8 x 14 MSE rotor 6 x 100 MSE rotor 6 x 100 MSE rotor SW 29 Sorvall rotor SS34
2
OF DENSITY GRADIENTS OF PERCOLL IN VARIOUS SOLVENTS AND CENTRIFUGES
Volume of Angle of centrifuge tube to tube axis (ml)
Solvent
GENERATED
Starting densities (g/cm”)
0.25M sucrose 1.05-1.16
Slope of the density gradient (g’ml-‘.cm-‘.lP) in the middle of the centrifuge tube after centrifugation at 10Gg x min.
23.5” 23.5” 25” 40 40”
13.5 13.5 94 6.5 6.5
0.15 0.25 0.25
M M
NaCl sucrose M sucrose 0.15M NaCl
1.05-1.14 1.08-1.16 1.05-1.12 1.06-1.13
3.5 7.5 2.0 1.1 6.0
90
13.5
0.15
M
NaCl
1.06-1.13
0.7
0.25 0.25 0.15 0.15 0.25 0.25 0.15
M
sucrose sucrose NaCl NaCl sucrose sucrose NaCl
1.08-1.16 1.05-1.16 1.05-1.14 1.06-1.13 1.08-1.12 1.08-1.16 1.06-1.13
3.0 1.8 7.0 5.5 1.5 0 6.0
18 29 29” 30 30 90 34”
100 14 14 100 100 38 50
M M M M M M
282
PERTOFT ET AL.
particles at low ionic strength is presumably due to a swelling of the polymer coat. The charges on the silica-surface, surrounded by a polymer network, could act as a polyelectrolyte gel. The low sedimentation rate in the absence of electrolytes leads to a slower generation of gradients during centrifugation (Table 2). Self-generated gradients of silica can be microheterogeneous, at least when formed in salt solutions of high ionic strength. Therefore, artifacts are observed when an homogeneous material is banded isopycnically in such gradients, because the material is separated into a number of closely situated, discrete zones (10). Such microheterogeneity has been observed also in other types of gradients, e.g., those made of albumin (11) and Ficoll(l2). In the experiments described in this paper the multiple banding could not be seen in pure Percoll or after addition of 0.25 M sucrose or 0.15 M NaCl. In preliminary runs performed at room temperature in phosphate-buffered Percoll, however, latex particles gave rise to several discrete bands. This phenomenon might be due to vibrations during centrifugation, leading to gradients with small discontinuities; however, this idea has not yet been fully explored. Users of Percoll should therefore be warned about drawing the conclusion that many distinct particle species have been separated when it is observed that the main bands are subdivided into a number of closely spaced sharp zones. At present there is no evidence to indicate that these artifacts interfere in any way with the separation of different species of particles. ACKNOWLEDGMENT This investigation (Project 3X-4).
has been supported
by the Swedish Medical
Research Council
REFERENCES 1. Mateyko, G. M., and Kopac, M. J. (1963) Ann. N. Y. Acad. Sci. 105, 185-286. 2. Pertoft, H., and Laurent, T. C. (1968) in Modem Separation Methods of Macromolecules and Particles (Gerritsen, T., ed.), Vol. 2, pp. 71-90, Interscience, New York. 3. Pertoft, H. (1969) Exp. Cell Res. 57, 338-350. 4. Wolff, D. A., and Pertoft, H. (1972) .I. Cell Biol. 55, 579-585. 5. Hjerten, S. (1967) Chromatogr. Rev. 9, 122-219. 6. Oster, G. (1965) Sci. Amer. 213, 70-77. 7. Dietz, E., and Hamann, K. (1976) Ang. Mukromol. Chemie 51, 53-60. 8. Iler, R. K. (1973) in Surface and Colloid Science (Matijevic, E., ed.), pp. 3-90, Wiley, New York. 9. Pertoft, H., and Laurent, T. C. (1977) in Methods of Call Separation (Catsimpoolas, N., ed.), Vol. 1, pp. 25-65, Plenum, New York. IO. West, C. M., and McMahon, D. (1976) Anal. Biochem. 76, 589-605. 11. Williams, N., Kraft, N., and Shortman-K. (1972) Immltnology 22, 885-899. 12. Bach, M. K., and Brashler, J. R. (1970) Exp. Cell Res. 61, 387-396.
BIOCHEMISTRY
88,271-282
(1978)
Density Gradients Prepared from Colloidal Silica Particles Coated by Polyvinylpyrrolidone (Percoll) HWKAN
PERTOFT,
TORVARD C. LAURENT, LENNART KAGEDAL
Institute ofMedical and Physiological Box 575, S-751 23 and Pharmacia
Chemistry, Universiry Fine Chemicals, Box
TORGNY
LAAs AND
of Uppsala, Biomedical 175 S-751 04 Uppsala,
Center, Sweden
Received May 23. 1977; accepted February 10, 1978 A new gradient medium (Percoll) for density gradient centrifugation of cells and subcellular particles is described. It consists of colloidal silica particles which have been firmly coated with a layer of polyvinylpyrrolidone. The particle population is polydisperse, and the average diameter is approximately 17 nm. Both the electrophoretic mobility of the particles and the conductivity of the solution are low, indicating a low surface charge. The colloid has a high solubility and forms clear solutions. It can be used in concentrations which give solution densities between 1.00 and 1.20 g/ml. The solutions have a low osmolality and can be mixed with electrolytes of physiological pH and ionic strength. The sedimentation properties of the particles are described. Density gradients can be formed by highspeed centrifugation. The colloid has been shown to be nontoxic for a number of cells and cell organelles.
Separation of cells and cell organelles by isopycnic gradient centrifugation is a potentially powerful technique, the application of which has been somewhat limited by the lack of suitable gradient materials. Most available substances form solutions of undesirably high osmolality or viscosity. Silica sols were first used by Mateyko and Kopac (1) for the “isopycnic cushioning” of cells from animals. Subsequently, Pertoft and co-workers [see Pertoft and Laurent (2)] established the general applicability of silica sols in density gradient centrifugation in a systematic investigation of the conditions for the separation of cells, viruses, and a variety of other particles. The applications have been somewhat limited, however, by the toxic effects of the silica. Pertoft (3) and Wolff and Pertoft (4) have described methods to prevent these effects, by adding polymers which are adsorbed on the surface of the silica particles. The presence of polymer in the solutions does, however, increase its viscosity, and it is also difficult to remove the polymer from the biological material after centrifugation. In this paper we describe the characteristics and uses of silica particles coated with a layer of polyvinylpyrrolidone (PVP). This product (Percoll) has many properties which renders it suitable for use in isopycnic separa271
0003-2697/78/0881-0271$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
272
PERTOFT
ET AL.
tion of cells and subcellular material. Water solutions of the product are of high density, low osmolality, and low viscosity. Generation of gradients is an automatic process occurring during centrifugation of the colloid. MATERIALS
AND METHODS
Colloidal silica ~01s. Pure colloidal silica, Ludox HS, was obtained from Du Pont de Nemours (Wilmington, Delaware). It was dialyzed repeatedly against distilled water before use. Colloidal silica coated with polyvinylpyrrohdone (Percoll) is a product of Pharmacia Fine Chemicals (Uppsala, Sweden). PEL-medium. PEL-medium was made according to Wolff and Pertoft (4) by mixing Ludox HS [12% (w/v)], polyvinylpyrrolidone (PVP) [5.3% (w/v); MW 40,000; from A. H. Thomas, Philadelphia, Pennsylvania], and Eagle’s minimum essential medium. Polyethylene glycol. Polyethylene glycol (PEG) (MW, 4000) was purchased from AB KEBO (Stockholm, Sweden). Chemical methods. Dry weight analysis of the particles was performed (heating to 100°C over phosphorus pentoxide in vacua to constant weight), and PVP content was calculated from nitrogen determinations according to Kjeldahl . Gel chromatography. Gel chromatography was performed at 4°C on Sepharose 4B using laboratory columns type K 15/90 from Pharmacia Fine Chemicals AB. A Uvicord III (LKB, Stockholm) was used to record the chromatograms by measuring transmission at 206 and 225 nm. Analytical ultracentrifugation. A Spinco Model E Analytical Ultracentrifuge equipped with schlieren optics was used to analyze the sedimentation behavior of silica particles. The experiments were performed in 12-mm double sector cells at a rotor speed of 12,000 rpm and a temperature of 20°C. Preparative ultracentrifugation. The following centrifuges were used to generate density gradients in Percoll: Beckman ultracentrifuges L4,L2-65B, and L50 equipped with the angle rotors 35, 40.2, and 65 and the swingout rotor 36; an MSE Super Speed 75 equipped with an 8 x 14 ml titanium angle rotor, (MSE), a 10 x loo-ml aluminum angle rotor (MSE), and a 6 x 38-ml aluminum swing-out rotor (MSE); an MSE 25 centrifuge with a 6 x 100 ml angle rotor; a Sorvall RC2-B centrifuge with an SS 34 angle rotor. Some experiments were performed in which sucrose to a final concentration of 0.25 M or sodium chloride to a final concentration of 0.15 M were added to the colloid. The samples were run for 10 to 60 min at speeds between 10,000 and 50,000 rpm and at a temperature of 20°C. The tubes were then fractionated by pumping Ludox HS to the bottom of the tube, and fractions obtained from the top were analyzed for density.
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273
Electron microscopy. A negative contrast technique was used, with 1% uranyl acetate in 0.2 M ammonium acetate (pH 4.6) as a contrasting agent. The specimens were examined in a Siemens Elmiskop I at a magnification of 80,000 using an operating voltage of 60 kV. A Zeiss particle analyzer was used to measure the distribution of particle diameters from the micrographs. Free zone electrophoresis. Free zone electrophoresis was carried out, as described by Hjerten (5). The runs were performed in 0.15 M sodium acetate (PH 4), 0.01 M phosphate buffer (pH 7.4), and 0.13 M NaCl, or 0.15 M Tris buffer (pH 9.0). The current was kept between 5 and 7 mA, and the voltage was between 300 and 1400 V. The glass tube was scanned every Iifth minute, and the absorbance at 280 nm and 320 nm was recorded. The migration of the samples was followed during 1 hr. Osmolality measurements. Osmolality measurements were performed in a freezing-point osmometer from Advanced Instrument Inc, Newton Highlands, Massachusetts). Conductivity. Conductivity was measured in a Conductivity Meter CDM 3 from Radiometer (Copenhagen, Denmark). Viscosity. Viscosity relative to water was measured in a KPG Ubbelohde Viscometer (JenaER Glas, Schott & Mainz, West Germany) at 20°C. Density. Density was measured in organic density columns (6) or in a densitometer (DMA 10 Digital Densimeter, Anton Paar, Graz, Austria). Refractive index. The refractive index was mesured in an Abbe refractometer. RESULTS
Chemical analysis of Percoll. Percoll is composed of solid silica spheres coated with PVP. The nitrogen content of the freeze-dried material is close to 1.50%, corresponding to 11.9% PVP. The PVP content varied between 11.4% and 12.4% in six different preparations. Gel chromatography. Gel chromatography was used to make a comparison between Percoll and a mixture of colloidal silica, PVP, and Eagle’s medium (PEL-medium (4)]. PVP was measured by its uv-absorption, and the silica was identified by density measurements on the fractions. In the Percoll preparation the PVP and silica material overlapped, indicating that PVP was firmly bound to the silica (Fig. 1). There was a small peak (amounting to between 1.2 and 3.2% as measured in different batches) close to the total volume, which was considered to be free PVP. When the main peak was rechromatographed, no material appeared at Vt. The PEL-medium showed a very different pattern (Fig. 1). A large uv-absorbing peak at the end of the chromatogram indicated that free PVP was present. There was some PVP bound to the silica, but the material showed a great heterogeneity.
274
PERTOFT ET AL. PEL
cq
“I
1
, PERCOLL
0
100
59
v, EFFLUEN
1
VOLUME
t vt (ml)
‘,
150
FIG. 1. Top: Gel chromatography of 2 ml of a mixture containing 12% (w/v) Ludox HS, 5.3% (w/v) PVP, and Eagle’s MEM [PEL-medium (4)] on a 1.5 x 85cm column of Sepharose 4B. The flow rate was 0.2 mUmin, and the eluent was 0.13 M NaCl + 0.01 M phosphate buffer (pH 7.4). PVP was analyzed by absorbance, and silica was analyzed by density measurements. Bottom: Gel chromatography of Percoll under the same experimental conditions.
It is known that polyethylene glycol can desorb polyesters which are adsorbed to silica surfaces (7). Therefore an experiment similar to that shown in Fig. 1 was carried out on Percoll with 2% PEG present in the eluant. This revealed no increase in the size of the small peak at the total volume, indicating that PEG had not been able to desorb PVP from silica. TABLE PROPERTIES
OF PERCOLL
AND DIALYZED
LUDOX
1 HS OF EQUAL
DENSITIES
(1.130
g/ml)
Colloidal solutions Property
Percoll”
Ludox HSb
Silica content (%, w/w) PVP content (%, w/w) Refractive index PH Viscosity (cP) Osmolality (mosM/kg of H,O) Conductivity (mS/cm)
20.0 2.7 1.3540 + 0.0005 8.8 k 0.1 9- 14 10 5 2 0.60 + 0.02
20.0 1.3485 + 0.0005 10 ” 0.2 2 + 0.2 20 + 2 1.50 k 0.02
U Figures are based on measurements of six batches of density, 1.130 + 0.005. * Figures are based on three batches of dialyzed Ludox HS. The values are not the same as those for undialyzed Ludox HS [compare Pertoft and Laurent, Ref. (9)].
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275
MATERIAL
Properties of Percoll solutions. Concentrated solutions of Percoll (up to a density of 1.2 g/ml) are colorless, slightly opaque liquids. They can be autoclaved (12O”C, 20 min) without any change in the properties. Table 1 shows a comparison between various properties of Percoll and those of a preparation of the original silica sol carefully dialyzed against distilled water. Although the PVP coat has no visible effect on the density of the solution, it can be seen that it does cause a
FIG. acetate
2. Electron at pH 4.6.
microscopy
of Percoll
particles
negatively
contrasted
with
1% uranyl
276
PERTOPT ET AL.
lowering of the osmolality and of the conductivity of the particles. This is supposedly due to a decrease in surface charge, which is also observed in electrophoresis. The relative viscosity of the solutions increases as a result of the polymer coating, but is still low for a density gradient medium. Electron microscopy. Percoll particles are shown in Fig. 2. They are not as aggregated, as is often found with pure silica particles [compare Fig. 1, Pertoft and Laurent (2)]. The colloid is polydisperse, as shown in Fig. 3. The mean particle diameter was determined to be 17.2 nm. The standard deviation of the size distribution was 2.3 nm. An identical result was obtained for a Ludox HS batch. Analytical ultracentrifugation. Both the Percoll and the Ludox particles sedimented as single peaks in the ultracentrifuge (Fig. 4). The concentration dependence of the sedimentation coefficient of Percoll is different from that of the uncoated particles (Fig. 5), there being a more rapid drop in the s02D,Wat low concentrations for coated than for uncoated particles. The sedimentation is also dependent on the ionic strength of the medium (see below). Electrophoresis. In free zone electrophoresis carried out at physiological ionic strength and pH 7.4, the mobility of the Percoll particles is only one tenth that of the original silica sol (Fig. 6A). The mobility of both Perco11 and Ludox HS varies with pH, and an isoelectric point of about 3 is indicated. This is in agreement with published data for colloidal silica (8). Variation of physical-chemical parameters with ionic strength. The viscosity of Percoll and of Ludox HS solutions, measured in water and in 0.15 M NaCl, is shown in Fig. 7. It is seen that the viscosity of
Diameter
(i-m)
FIG. 3. Percoil (-) and Ludox HS (-----) particles were examined in the electron microscope (magnification 68,000X), and by utilizing a particle analyzer on the photographic plates, the number of particles and the particle diameters were measured. The diameter was 17.3 nm for Percoll and 17.2 nm for Ludox HS. The standard deviation was 2.3 nm in both cases.
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277
MATERIAL
A
FIG. 4. Analytical ultracentrifugation of coated and uncoated colloidal silica at a rotor speed of 12,000 rpm and a concentration of 3%. The wedge cell (upper curve) has 0.15 M NaCl as solvent and the standard cell (bottom curve) has water as solvent. (A) Ludox HS; and (B) Percoll. Pictures were taken with a bar angle of 60” after 16 min at full speed.
Percoll solutions is strongly dependent on ionic strength, in contrast to that of Ludox HS. The sedimentation coefficient of Percoll was measured in sodium chloride of various ionic strengths (Fig. 5). In both dilute and concentrated solutions of Percoll, the particles sediment slower at low PERCOLL
I 0
LUDOX-HS
I
70 CONCENTRATION
20
0 OF
10 COLLOIO
M I%.
w/wl
FIG. 5. The sedimentation constant of Percoll and of Ludox HS as a function of concentration. The runs were made in 0.15 M NaCl (0 0); 0.01 M NaCl (0 0); 0.001 M NaCl (m n ); and distilled water (0 0).
278
PERTOFT
T/ME
ET AL.
fsec
. IO-?,
A
PH
FIG. 6. (A) Free zone electrophoresis of Percoll (0 0) and Ludox HS (0 0). A 10-~1 sample of a particle suspension with a density of 1.023 g/ml was applied in each run. The experiments were carried out in 0.01 M phosphate buffer (pH 7.4) + 0.13 M NaCI. Voltage, 300 V; current, 6 mA; and temperature of the cooling water, 10°C. Scans were made regularly up to 4200 set, and the migration distance versus time is shown. (B) The mobility of particles of Ludox HS and of three different batches of Percoll was measured in several runs at pH 4 in 0.15 M sodium acetate buffer; at pH 7.4 in 0.01 M phosphate buffer + 0.13 M NaCI; and at pH 9 in 0.15 M Tris buffer. Experimental details are described in (A).
ionic strength than they do at physiological salt concentration. This phenomenon is more pronounced for Percoll than for Ludox HS. Gel chromatography performed as described in the legend to Fig. 1 but with distilled water as eluent resulted in a displacement of the Percoll peak toward the void volume. Pure Ludox HS cannot be chromatographed, as it adsorbs to the Sepharose. The results from viscosity measurements, gel chromatography, and sedimentation studies indicate that the Percoll particles occupy a larger hydrodynamic volume when the ionic strength is decreased. Free zone electrophoresis was performed in 0.02 M phosphate buffer (pH 7.4) and in the same buffer with the addition of 0.03, 0.13, or 0.28 M NaCl. There was an increase in mobility with
A NEW DENSITY
CONCENTRATION
GRADIENT
OF
COLLOID
MATERIAL
279
(%.‘+‘/w)
FIG. 7. The viscosity of Percoll and Ludox HS solutions as a function of concentration. The measurements were made in distilled water (0 0) and in 0.15 M NaCl(0 __ 0).
decreasing ionic strength (from 1 x 10-j to 5 x 1O-5 cm2.sec-’ *V-l), but the experiments at low salt concentration were technically difficult to perform. It was not possible to compare the effect of ionic strength on Percoll with that on pure colloidal silica at ionic strengths above 0.2 M because of the instability of the latter (2). Generation of density gradients of Percoll. Formation of density gradients of Percoll can be performed by conventional methods, but it can also be done by high-speed centrifugation. The gradients are the result of sedimentation of the colloidal particles. Because the material is polydisperse (Figs. 2 and 3), the individual particles sediment with different speeds, resulting in a smooth concentration gradient. Examples of gradients generated in the centrifuge are shown in Fig. 8A. These examples also demonstrate that the slope of the gradients which are formed is essentially independent of the starting density of Percoll. It could also be shown empirically that the slope of the gradient generated is approximately linearly related to the time and g-force of the centrifugation (Fig. 8B). Other factors which influence the shape of the gradients are the size of the centrifuge tubes, the geometry of the rotor, and the solvent in which Percoll is dissolved. Table 2 summarizes a large number of experiments in which gradients were generated under various experimental conditions, and it may serve as a guide in selection of centrifugation conditions. DISCUSSION
The present work is based on the earlier observation that colloidal silica solutions can be used for density gradient centrifugations and that the toxic effects of the silica can be inhibited by adding polymers such
280
PERTOFT ET AL.
M DISTANCE
A
B
FROM
g-FORCE
THE
MENISUS
I MIN
B (cm)
I lo5
FIG. 8. Development of density gradients during centrifugation of Percoll solutions. (A) Different concentrations of Percoll dissolved in 0.25 M (isotonic) sucrose were centrifuged in an MSE 8 x 14 ml rotor at an average g-value of 60,000 for 30 min (A A) and 60 min (0 - - - 0). The density gradients formed are shown. The densities of the solutions before the runs are given on the right-hand side. The slopes of the gradients were recorded in the middle of the tubes [see Table 2 and (B)]. M and B denote the menisci and bottoms of the centrifuge tubes. (B) The slope of the density gradient in the middle of centrifuge tubes was recorded after centrifugation of Percoll in 0.25 M sucrose for various times at varying centrifugal forces in two different rotors. The starting density of the Percoll solution varied between 1.05 and 1.16 g/ml, but the gradients which developed show little dependence on the starting density [see (A)]. The slope of the gradient increases approximately linearly with the amount of centrifugation (g-force x time). The runs were made in an MSE rotor (14-ml tubes, angle 29”) (---) and Beckman rotor 6.5 (135ml tubes, angle 23.5”) (-).
as polyethylene glycol, dextran, and polyvinylpyrrolidone (3,4). The polymers were adsorbed to the silica in the presence of an excess of free polymer [see, e.g., Ref. (2)]. The presence of excess polymer in the solution has, however, many drawbacks.
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GRADIENT
281
MATERIAL
Percoll is essentially free of unbound PVP, as judged from the gel chromatograms. The PVP coat seems to be firmly attached to the silica surface and cannot be desorbed by polyethylene glycol. The thickness of the PVP coat can be calculated from the following data: The PVP content is analyzed to be 11.9%, the densities of silica and of PVP are 2.2 and 1.2 g/ml, respectively, and the diameter of the silica particles is 17.2 nm. The value of 0.6 to 0.7 nm so calculated is close to monomolecular. No difference in the diameter of coated and uncoated silica could be detected using the electron microscope, but it is possible that the contrasting agent penetrates the coat. If the PVP coat contains water, it would be thicker than 0.7 nm. The coating of the silica gives it many advantages over the pure silica sol. The colloid is more stable, especially in physiological salt solutions; it has a low osmolality, apparently because of a decreased or masked surface charge; it has a low toxicity toward cells; and it can be used to fractionate cell organelles such as lysosomes and viruses (9). Despite a low surface charge, as judged from electrophoresis, the viscosity and sedimentation of Percoll are more dependent on ionic strength than they are for pure silica (Figs. 5 and 7). The apparently larger hydrodynamic volume of the TABLE CHARACTERIZATION
Type of rotor Beckman rotor 6.5 Beckman rotor 65 Beckman rotor 35 Beckman rotor 40.2 Beckman rotor 40.2 Beckman rotor SW 36 MSE rotor 10 x 100 MSE rotor 8 x 14 MSE rotor 8 x 14 MSE rotor 6 x 100 MSE rotor 6 x 100 MSE rotor SW 29 Sorvall rotor SS34
2
OF DENSITY GRADIENTS OF PERCOLL IN VARIOUS SOLVENTS AND CENTRIFUGES
Volume of Angle of centrifuge tube to tube axis (ml)
Solvent
GENERATED
Starting densities (g/cm”)
0.25M sucrose 1.05-1.16
Slope of the density gradient (g’ml-‘.cm-‘.lP) in the middle of the centrifuge tube after centrifugation at 10Gg x min.
23.5” 23.5” 25” 40 40”
13.5 13.5 94 6.5 6.5
0.15 0.25 0.25
M M
NaCl sucrose M sucrose 0.15M NaCl
1.05-1.14 1.08-1.16 1.05-1.12 1.06-1.13
3.5 7.5 2.0 1.1 6.0
90
13.5
0.15
M
NaCl
1.06-1.13
0.7
0.25 0.25 0.15 0.15 0.25 0.25 0.15
M
sucrose sucrose NaCl NaCl sucrose sucrose NaCl
1.08-1.16 1.05-1.16 1.05-1.14 1.06-1.13 1.08-1.12 1.08-1.16 1.06-1.13
3.0 1.8 7.0 5.5 1.5 0 6.0
18 29 29” 30 30 90 34”
100 14 14 100 100 38 50
M M M M M M
282
PERTOFT ET AL.
particles at low ionic strength is presumably due to a swelling of the polymer coat. The charges on the silica-surface, surrounded by a polymer network, could act as a polyelectrolyte gel. The low sedimentation rate in the absence of electrolytes leads to a slower generation of gradients during centrifugation (Table 2). Self-generated gradients of silica can be microheterogeneous, at least when formed in salt solutions of high ionic strength. Therefore, artifacts are observed when an homogeneous material is banded isopycnically in such gradients, because the material is separated into a number of closely situated, discrete zones (10). Such microheterogeneity has been observed also in other types of gradients, e.g., those made of albumin (11) and Ficoll(l2). In the experiments described in this paper the multiple banding could not be seen in pure Percoll or after addition of 0.25 M sucrose or 0.15 M NaCl. In preliminary runs performed at room temperature in phosphate-buffered Percoll, however, latex particles gave rise to several discrete bands. This phenomenon might be due to vibrations during centrifugation, leading to gradients with small discontinuities; however, this idea has not yet been fully explored. Users of Percoll should therefore be warned about drawing the conclusion that many distinct particle species have been separated when it is observed that the main bands are subdivided into a number of closely spaced sharp zones. At present there is no evidence to indicate that these artifacts interfere in any way with the separation of different species of particles. ACKNOWLEDGMENT This investigation (Project 3X-4).
has been supported
by the Swedish Medical
Research Council
REFERENCES 1. Mateyko, G. M., and Kopac, M. J. (1963) Ann. N. Y. Acad. Sci. 105, 185-286. 2. Pertoft, H., and Laurent, T. C. (1968) in Modem Separation Methods of Macromolecules and Particles (Gerritsen, T., ed.), Vol. 2, pp. 71-90, Interscience, New York. 3. Pertoft, H. (1969) Exp. Cell Res. 57, 338-350. 4. Wolff, D. A., and Pertoft, H. (1972) .I. Cell Biol. 55, 579-585. 5. Hjerten, S. (1967) Chromatogr. Rev. 9, 122-219. 6. Oster, G. (1965) Sci. Amer. 213, 70-77. 7. Dietz, E., and Hamann, K. (1976) Ang. Mukromol. Chemie 51, 53-60. 8. Iler, R. K. (1973) in Surface and Colloid Science (Matijevic, E., ed.), pp. 3-90, Wiley, New York. 9. Pertoft, H., and Laurent, T. C. (1977) in Methods of Call Separation (Catsimpoolas, N., ed.), Vol. 1, pp. 25-65, Plenum, New York. IO. West, C. M., and McMahon, D. (1976) Anal. Biochem. 76, 589-605. 11. Williams, N., Kraft, N., and Shortman-K. (1972) Immltnology 22, 885-899. 12. Bach, M. K., and Brashler, J. R. (1970) Exp. Cell Res. 61, 387-396.