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J. Biochem. Biophys.

Methods 44 (2000) 130


www.elsevier.com / locate / jbbm
Review
Fractionation of cells and subcellular particles with
Percoll q


Hakan Pertoft*
Department of Medical Biochemistry and Microbiology, Unit of Biochemistry, University of Uppsala BMC,
Box 575 Uppsala, SE-75123, Sweden

Accepted 15 June 1999

Abstract

At present, centrifugation is the most common method for separation and isolation of cells and
subcellular particles. The technique can be used for a wide range of applications. During latter
years it has become obvious what a powerful method density gradient centrifugation is, especially
when used in conjunction with sensitive assays or clinical treatments. The most active areas for
use of density gradient centrifugation include puri cation for in vitro fertilization of sperm of both
human and bovine origin, isolation of cells for cell therapy of patients receiving chemo- and
radiation therapy and basic research both on cellular and subcellular levels. These treatments and
investigations require homogeneous populations of cells and cell organelles, which are undamaged
after the separation procedure. Percoll, once introduced to reduce convection during centrifugation,
has proved to be the density gradient medium of choice since it ful lls almost all criteria of an
ideal density gradient medium. Recently good results have also been obtained after silanization of
colloidal silica particles, e.g. BactXtractorE. The latter medium has proved to be useful in
recovery of microorganisms from food samples free of inhibitors to the Polymer Chain Reaction
(PCR). The separation procedures described for Percoll in this review seem to be applicable to any
cells or organelles in suspension for which differences in size or bouyant density exist.
Furthermore, since Percoll media are inert, they are well suited for the separation of fragile
elements like enveloped viruses. 2000 Elsevier Science B.V. All rights reserved.

Keywords: Cells; Centrifugation; Density gradients; Percoll; Separation; Subcellular particles

q
Percoll is a registered trademark of Amersham Pharmacia Biotech AB, Box 582 Uppsala, Sweden.
*Tel.: 1 46-18-471-4442; fax: 1 46-18-471-4673.
E-mail address: hakan.pertoft@medkem.uu.se (H. Pertoft)

0165-022X / 00 / $ see front matter 2000 Elsevier Science B.V. All rights reserved.
PII: S0165-022X( 00 )00066-X
2 H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30

1. Introduction

1.1. Density gradient centrifugation in general

Separation of cellular particles from biological uids and tissues is of importance both
in clinic practice and in basic research. Also, studies on cultured cells and subcellular
components, e.g. lysosomes, membrane vesicles, mitochondria, nuclei and ribosomes
often require highly puri ed preparations.
The isolated cells and microorganisms must be unimpaired regarding viability and
biological function after the separation process. It is important that the separation and
puri cation procedure is as harmless as possible and that no interfering substances are
introduced during the process.
Density gradient centrifugation in a de ned medium ful lls these requirements. Most
of these media have been developed since the 1960s (see Section 1.2). Depending on the
kind of separation required, centrifugation conditions such as centrifugal force, centrifu-
gation time and type of centrifuge rotor can be varied.
Centrifugation has been used in chemical laboratories since 1852 [1]. For diagnostic
purposes it was introduced to determine the volume of red cells in blood (hematocrite)
[2]. After the publication of the classical monograph by Svedberg and Pedersen [3] the
analytical applications centrifugation multiplied, and there were numerous practical
re nements and extension of theory. Initially uniform solutions were studied in moving-
boundary systems using sector-shaped cells or zonal rotors. In these systems particles
moved in radial directions and there were no problems with convection. Svedberg et al.
characterized a large number of proteins by analytical ultracentrifugation in convection-
free systems.
To prepare and purify material in cylindrical centrifuge tubes in swing-out rotors has
been more dif cult as the gravitational force works in the radial direction and introduces
convection. The material concentrates at the tube walls and is diluted in the center of the
tube. This results in a radial convection and material will start to stream up in the center
of the tube. To prevent this effect, Pickles developed density gradients to stabilize the
moving boundaries during sedimentation [4]. The resulting convection in the tubes was
reduced to levels of equal density. Brakke later layered the suspension to be fractionated
on top of a solution having an increased density along the tube [5].
The density gradient method of separation has several advantages. There is no mixture
of materials beneath the sample zone as in normal differential centrifugation. During
centrifugation all cells or particles of the same size, shape and density sediment as
separate zones without convection [6]. Furthermore, a complete separation of several
components in a mixture according to size (rate zonal centrifugation) or density
(isopycnic centrifugation) can be performed by varying sedimentation time, g-force and
the range of the density gradient interval.

1.2. Media for density gradient centrifugation

There are a number of characteristics of the ideal density gradient material [7]. Various
gradient media have been developed for speci c applications.They should, if possible,
H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30 3

not alter the cells or particles to be separated and should provide a useful density range
for separation. The obstacles to overcome are toxicity, osmotic pressure changes and
penetration into particles [7].

1.2.1. Sucrose and salts


The main disadvantages of sucrose solutions are some of their physico-chemical
properties. Sucrose solutions in the concentration range used have a high osmolality
(Fig. 1). and are viscous. Cells and subcellular particles, which are osmotically sensitive,
will band at a density which differ from their physiological density. Furthermore, its low
molecular weight (342) makes it possible for sucrose to penetrate into cells and
enveloped particles. Also other media composed of low molecular weight solutes, e.g.
CsCl, K-tartrate and NaBr, display high osmotic pressure and pass into cells, making
them less suitable for general applications.

1.2.2. Polysucrose
A synthetic polymer of sucrose (Ficoll; Amersham Pharmacia Biotech, Uppsala,
Sweden) was introduced early to overcome the problem of high osmotic pressures [8].
However, Ficoll of molecular weight 400,000 also gives measurable osmotic gradients
with increasing concentrations, and this has to be compensated for by a saltgradient to
keep the iso-osmotic conditions throughout the centrifuge tube (Fig. 1). Also a natural
polymer of glucose (dextran; Amersham Pharmacia Biotech, Uppsala, Sweden) with
molecular weights of 10 5 10 6 has been used to fractionate membranous vesicles and
cells [9]. Unfortunately these solutions of dextran are too viscous for convenient
handling.

Fig. 1. Variation in osmolality with concentration for sucrose of molecular weight 342 and Ficoll of molecular
weight 400,000 in water. Percoll, molecular weight 6 3 10 6 , was adjusted with saline to an osmolality of 300
mOsm before the analysis.
4 H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30

1.2.3. Iodinated compounds


Iodinated compounds developed for use as X-ray contrast media are also widely used
as centrifugation media, e.g. Hypaque, Urogra n, Metrizoate, Isopaque, Metrizamide,
Nycodenz, Optiprep, etc. [7]. In these solutions cells band isopycnically without being
subject to the high osmotic stress of sucrose gradients.

1.2.4. Mixed gradients


In 1968 Arne Byum published a method to isolate mononuclear cells from human
peripherial blood based on a mixture of Ficoll and an iodinated compound [10]. Similar
media are now commercially available, e.g. Ficoll-Paque, Histopaque, Histoprep,
Lymphoprep, etc. [7]. However, because of the low molecular weight of the iodinated
components in X-ray contrast media, and even though they are nonionic, they are able to
diffuse into cells and cell organelles and make the buoyant densities unphysiological.

1.2.5. Colloidal silica


This review does not provide a thorough scrutiny of the various gradient media
available, but describes in more detail the practical use of colloidal silica as a gradient
medium. Its unique combination of properties makes colloidal silica useful for most
applications in cell science.
The use of colloidal silica was rst reported in 1959 by Mateyko and Kopac [11].
They reported the osmotic pressure effects, ability to separate cells, permeation into
particles and solubility in aqueous solutions of various density gradient media. Of all
substances tested, none came closer to providing all the desired characteristics than
colloidal silica. However, it was found that a pure silica sol was toxic to cells and caused
hemolysis of red blood cells [12]. At the time when polysaccharides were introduced to
stabilize colloidal silica gradients they were also found to inhibit toxic effects of the
silica [13,14]. Further development of modi ed colloidal silica has been described [15].
The introduction of adsorbed polymers to silica particles to obtain iso-osmotic, pH-
neutral and high density solutions led to the introduction of Percoll in 1977 (Amersham
Pharmacia Biotech, Uppsala, Sweden).
6
Percoll has a mean molecular weight of 6 3 10 and and an osmolality , 20 mOsm at
a density of about 1.13 g / ml. Physiological salt can be added to Percoll to keep the
osmolality constant at the density intervals used for cells and cell particles (Fig. 1). The
medium was adopted early by several research teams, who published good results on
isolation of tissues, cells and cellorganelles [16]. After these early publications there was
a rapid growth in the use of Percoll, especially in cell separations.

2. Physico-chemical properties of Percoll

The silica in Percoll is a sodium-stabilized colloid, which is polydisperse with particle


diameters between 10 and 30 nm [16]. To protect the cells from the toxic action of the
H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30 5

Fig. 2. Properties of Percoll particles. The dissociated silica colloid particle is covered by a layer of
polyvinylpyrrolidone (PVP). The molecular weight is 6 3 10 6 and the dry particle has a diameter of 2122 nm.
The charges on the silica-surface, surrounded by a PVP network, allow the particle to act as a polyelectrolyte.
In water the hydrated particle with PVP swells to a diameter of 35 nm (A). At higher ionic strength an almost
complete monolayer of PVP is formed outside the particle which has a diameter of 30 nm (B).

colloidal silica, polyvinylpyrrolidone (PVP) is coated on the particle (Fig. 2). In water
the PVP is expanded by the counter ions to silica and and the average diameter is 35 nm
(Fig. 2A). At higher ionic strength the PVP is closely attached to the silica particles
which have an average diameter of 30 nm in 0.15 M NaCl (Fig. 2B). This indicates that
there is a layer of hydration on the particles which is more pronounced at lower ionic
strengths [17]. Also the viscosity of Percoll is a function of the ionic strength, and is
lower in saline solutions compared to water [18]. This has as an effect that self-
generated gradients are created much more rapidly in 0.15 M NaCl than in iso-osmotic
(0.25 M) sucrose.
Electron microscopic analysis of the particles in gradients generated by high speed
centrifugation in an angle-head rotor shows that the Percoll at the bottom of the tube is
considerably enriched in aggregates and larger particles (Fig. 3A). At the top of the
gradient the particles are smaller in size and more diluted (Fig. 3B). This is the same
result as is obtained during any differential centrifugation, i.e. the only pure homoge-
neous fraction obtained is in the supernatant, while the pellet consists of a mixture of
pelleted material together with material which was already present at the bottom of the
tube prior to centrifugation.
The gradients of Percoll become progressively steeper with centrifugation time and
g-force (Fig. 4). Prolonged centrifugation at high g-forces results in a complete
sedimentation of the colloid and the formation of a gel at the bottom of the tube.
Therefore, material to be separated having sedimentation coef cients of , 90 S,
corresponding to the s 20,w -value of Percoll in water [18], will oat on top of the Percoll
gradient.
6 H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30

Fig. 3. Electron microscopy of Percoll particles negatively contrasted with 1% uranyl acetate at pH 4.6. A
50% Percoll solution in PBS was centrifuged for 20 min at 20,000 3 g in an angle rotor. A bottom fraction and
top fraction was analyzed on micrographs with a Zeiss particle analyzer. The results showed that the mean size
and number of particles was increased in the bottom fraction (A) compared to the top fraction (B).
Magni cation 3 68,000.
H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30 7

Fig. 4. Gradient formation in Percoll by centrifugation in an angle rotor. The starting density was 1.065 g / ml
(50% Percoll in 0.15 M NaCl). Running conditions: 40,000 3 g for 30 min (1); 60 min (2); and 90 min (3) at
208C. Gradient density was monitored by means of colored Density Marker Beads.

3. Experimental methods

3.1. Preparing the gradient medium.

There is substantial confusion in the literature about the true concentration of Percoll
used in some methods, where it was not made clear whether 100% was an undiluted
Percoll as it came in the bottle in pure water from the manufacturer, or 9 / 10 prediluted
isotonic Percoll solution re-de ned as 100%. This latter mixture is called 100% Percoll
in the following. In order to use Percoll to prepare a density gradient, the osmolality of
Percoll from the bottle must usually rst be adjusted with saline or cell culture medium
to make Percoll isotonic with the biological material to be separated. It is well known
that the osmotic pressure varies in various organs and body uids and also varies in
different species [19,20]. The osmolarity of human serum is 288 mOsm, which is
equivalent to a freezing depression of 2 0.5358C [21]. Adding 9 parts (v / v) of Percoll to
1 part (v / v) of 10 3 concentrated phosphate buffered saline (PBS from Gibco BRL) is a
simple way of preparing a stock hypertonic (354 mOsm) Percoll solution of pH 7.4 [22].
The density of this stock isotonic Percoll solution (100% Percoll) will be 1.123 g / ml
and further dilutions to lower densities with 1 3 PBS gives a linear correlation between
concentration and density (Fig. 5). To obtain a stock solution of about 300 mOsm, a
quick procedure is to add 1 part of 10X concentrated PBS to 12 parts of Percoll [22].
Alternatively add 0.8 g NaCl per 100 ml of Percoll to obtain an osmolality of about 300
mOsm and adjust the pH with 6 N HCl to get a pH of 7.4. Osmolality, density and pH of
all stock solutions should, however, be checked routinely.

3.2. Sample loading

Sample suspensions can be applied on Percoll gradients in three different ways:


8 H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30

Fig. 5. The density of Percoll solutions as a function of concentration. The stock isotonic Percoll was prepared
by adding 1 part of ten times concentrated phosphate buffered saline (10 3 PBS) to 9 parts of Percoll. This
mixture is called 100% Percoll. Further dilutions were performed with phosphate buffered saline (PBS). The
density of the solutions was measured by pykonometry.

3.2.1. Mixing of sample and gradient medium


Isotonic Percoll is mixed with the sample and the gradient is formed in situ by high
speed centrifugation [15]. Cells and particles will band at their bouyant density during
the centrifugation.The technique is convenient for puri cation of large viruses at
20,00030,000 3 g and a centrifugation time of 2030 min [23].
For cells it is also possible to mix the sample with Percoll prior to centrifugation and
keep it in a horizontal position (Fig. 6A). During centrifugation in a swinging-out rotor
with the tube in a vertical position, at low speed, the cells starts to move in the direction
of gravity. Simultaneously a gradient is formed by the sedimentation of Percoll particles.
Cellular particles will separate according to size or density depending on centrifugation
time and g-force used (Fig. 6B). The separated fractions are close to each other during
the separation procedure in the vertical position and at rest in the horizontal position as
well. When the tube is placed on the laboratory bench, however, the cell bands reorient
which leads to increased distances between the fractions (Fig. 6C). The same
phenomenon occurs in a vertical rotor used for high speed centrifugation, but these
rotors are not suitable for Percoll because the gradient is formed too rapidly [7].

3.2.2. Sample layered on top of a density gradient


The technique to layer the sample on top of a preformed gradient reduces contamina-
tion by cell debris, etc., which remains in the top layer after separation. Puri ed fractions
sediment to their bouyant density or according to their size.
If the sample is layered on top of a density gradient and reoriented to a horizontal
H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30 9

Fig. 6. In a swing-out rotor and with the tube in a vertical position, the sedimentation path length, the running
time and the g-force can be signi cantly shortened. (A) The sample is mixed with 50% v / v Percoll in 0.15 M
NaCl in a 50 ml Falcon tube and placed in horizontal position in a swing-out rotor; (B) during centrifugation at
800 3 g (2000 rpm at 18 cm radius) the tube comes to a vertical position and a stabile gradient is formed with
isolated fractions close to each other; (C) after centrifugation the tube is placed on the laboratory bench, which
results in an increased distance between fractions depending on the length of the tube.

position, the sample is distributed over the whole length of the tube. This technique
reduces centrifugation time and centrifugal force as described in Fig. 6.

3.2.3. Underlayering of sample


The sample can also be mixed with concentrated Percoll and layered below a
preformed gradient. A lighter solution of Percoll may also be layered on top of the
10 H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30

sample and during centrifugation a gradient will form if the run is performed at a high
g-force [24]. One advantage with this technique is that both aggregates and low
molecular substances remain in the bottom fraction after centrifugation and only pure
cells or particles oat to their isopycnic level (Fig. 7).

3.3. Gradient formation

Three different systems can be used to form the gradients: step density gradients;
self-forming gradients and preformed continuous gradients (Fig. 8).

3.3.1. Step gradients


Step or discontinuous gradients often give good separation of cells, but are not as
sensitive or selective as a continuous gradient. Discontinuous gradients are especially
useful when separating only a few different cell types of known density.
The gradient is prepared by layering one or several Percoll solutions of different
concentration of Percoll along the centrifuge tube (Fig. 9). The cells will be concentrated
at the different interfaces as visible bands. One drawback with the technique is that
contaminating cells often will be trapped at the wrong interfaces, i.e. the purity of
fractions is often low with this type of gradient formation.
It often happens that even if the intention has been to use discontinuous gradients,
these became continuous [25]. The reason is that if a large volume of e.g. blood is
layered on top of Percoll and centrifuged, the cells pull serum through the Percoll layer
and form a continuous gradient. The gradient formed will depend on the volume of

Fig. 7. Underlayering of sample. The cellsuspension is mixed with concentrated Percoll and layered below a
preformed gradient (A). After centrifugation at low speed for a short period of time cells start to oat (B). In
the bottom fraction, cell debris remain together with heavy aggregates in rate zonal experiments.
H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30 11

Fig. 8. Gradient formation. Percoll from the bottle is rst diluted with concentrated saline or salt added as
powder to make the working stock solution. This solution is then further diluted with physiological buffers
according to need. Preformed gradients can be generated by centrifugation or by use a gradient-mixing device.
The self-forming gradients are always formed by high speed centrifugation and not by diffusion.

blood applied, the hematocrite and the original volume and density of the Percoll
solution. Scriba et al. [26] found that 45 3 10 7 leukocytes in a volume of 33 ml could
be overlaid 14 ml 60% Percoll in a 50 ml Falcon tube. The monocytes were obtained
with a recovery of 97% at the interphase and a viability of . 99% of this type of
gradient [26].

3.3.2. Continuous gradients


Normally, continuous gradients are prepared employing a gradient-mixing device.
Several manufacturers produce various devices for making gradients of various shape,
e.g. ISCO Inc., Lincoln, Nebraska or Beckman Instrument, Fullerton, CA. One can
prepare linear, convex, concave or S-shaped gradients, depending on the need and
availability of equipment [27]. A linear gradient is prepared by introducing the denser
liquid into a vessel containing the less dense liquid, where the two liquids are mixed on
a magnetic stirrer. The resulting mixture ows out of this latter vessel through a tubing
that leads to the bottom of the centrifuge tube. It is important to avoid air bubbles at the
end of gradient formation [7].
Continuous gradients of Percoll are also formed by freezing and thawing of the
solution. It is an exclusion of solute during freezing followed by a oatation of the
lighter ice which forms the gradient. Unfortunately these gradients cannot be used for
cells, sperm or bacteria if the aqueous thawed gradients have not been mixed with
diluents containing double-strength salt [28].
S-shaped Percoll gradients can be formed under high forces of gravity when the
Percoll particles sediment as described above. When self-forming gradients of Percoll are
used, one should preferably choose a density at the center of the tube which is in
between the density of the particles to be separated. The length and volume of the tube,
type of rotor and desired shape of the gradient, volume of the tube, type of rotor, and
density of the gradient determine the time and speed to be used for forming the gradient
[18]. We have found that, with self-forming Percoll gradients, the central linear portion
12 H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30

Fig. 9. Density gradient maker. A syringe is connected to a two-way stopcock, which is connected to a needle
which reaches the bottom of the centrifuge tube. With the stopcock in the off position, the less dense Percoll
solution is poured into the syringe. Then the stopcock is closed and the next layer lled in the same way. It is
important to avoid air bubbles when preparing a density gradient.

of the gradient is nearly at when formed in a swing-out rotor and is not as satisfactory
as that formed in an angle head rotor. As described above, Fig. 6, we have found that
swing-out rotors are more useful to reorientate Percoll solutions if the tubes are in
vertical positions. Under such conditions S-shaped gradients are generated already at
2000 rev / min and a centrifugation time of 20 min (Fig. 10).
Osmocentrifugation of Percoll solutions is a new technique to prepare continuous
self-generated density gradients at 2002000 3 g during 10-30 min of centrifugation
[29]. The technique is simple, but needs a special dialysis cell. Under conditions of unit
gravity, there is a net ow of solvent molecules across a semi-permeable membrane by
osmosis into the Percoll solution. By centrifuging at low speeds (2002010 3 g),
increased pressure within the compartment containing Percoll leads to a degree of
reverse osmosis which varies at different depths. The overall effect is that approach to
sedimentation equilibrium is observed within hours. In our laboratory we have not found
H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30 13

Fig. 10. Gradient development in a swing-out rotor with the tubes in vertical positions. Stock isotonic Percoll
was diluted with isotonic saline to density 1.040 g / ml; 1.056 g / ml; and 1.100 g / ml. Centrifugation was
performed for 20 min at 2000 rpm (800 3 g at 18 cm radius) with the tubes in a vertical position as described
in Fig. 6. Density Marker Beads were mixed with the gradient medium and during centrifugation the beads
moved in the gravitational direction. After centrifugation the tubes were carefully placed on the laboratory
bench and the distances from the bottom to each bead was measured. (ss) density 1.04 g / ml; (hh)
density 1.056 g / ml; and (nn) density 1.100 g / ml.

the technique especially useful because of dif culties in assembling and opening the
dialysis cell. Furthermore, the technique is limited to cell separation.

3.4. Centrifugation parameters

Large particles sediment more quickly than small particles. However, some small, but
very dense particles might sediment faster than larger but very light particles. This fact
has been described in several reviews [7,30]. The sedimentation occurs of course only if
the density of the uid is less dense than the density of the particles. The Percoll density
barrier is a solution of a density that allows some particles to pass through it and others
to be prevented from entering it. A small dense particle initially sediments less rapidly
than a larger particle of low density. The large particles reach their position of bouyant
density early, while the small, dense particles slowly pass through the large particle zone
and band at another level in the gradient. For convenience, particles are characterized by
their sedimentation coef cients measured in water at 208C, s 20,w . If the particles are
regarded as spherical and the diameter and their density are known, it is possible to
calculate sedimentation coef cients [30]. The buoyant densities can be determined in
Percoll gradients and are in the range 1.051.20 g / ml. The graph in Fig. 11 shows the
relationship between sedimentation coef cients and particle diameters of such particles.
14 H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30

Fig. 11. Particle diameters related to sedimentation coef cients (s 20,w ). This graph is valid for spherical
particles or cells, where the bouyant density in Percoll is 1.05 g / ml (jj); 1.10 g / ml (mm); and 1.20 g / ml
(dd).

3.5. Fractionation

Harvesting of isolated cells or particles after centrifugation can be carried out by any
standard technique. One technique that we found simple and effective for the collection
of separated cells is shown in Fig. 12. By introducing undiluted Percoll under the
gradient, the contents are forced up and out of the collection tube. It is preferable to have
a stopper, which is concave at the outlet tube end to avoid mixing of fractions. Piercing
the tube at the bottom is not recommended because of the mixing of fractions if the tube
bottom is not conical. Similarly, to take fractions from the top of the tube by the use of a
pipette contaminates fractions from the top with fractions lower down in the tube.
If an inner column in the form of disposable tip has been used for the formation of the
gradient inside a centrifuge tube, the inner column can be closed with a rubber cap and
removed before the sediment is harvested in the centrifuge tube [31]. One of the most
accurate and sensitive gradient fractionation systems has recently been developed at
BioComp Instrument, Inc., Fredericton, NB, Canada.

3.6. Density analysis

Density measurements of isolated fractions is best performed by use of Density


Marker Beads (DMB), Amersham Pharmacia Biotech, Uppsala, Sweden. These are
color-coded Sephadex beads, which are modi ed to have a speci ed density in gradients
of Percoll [32]. By centrifuging a mixture of these beads in a separate tube and recording
the level at which they band, the gradient can be de ned and compared to the position of
H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30 15

Fig. 12. Fractionation of gradients. The gradient is displaced upwards towards an unloading cap by
introducing a dense displacing liquid (100% Percoll) at the bottom of the tube using a pump. The displaced
gradient may be monitored optically by passage through a spectrophotometer ow cell before collection into
series of fractions.

visible cellbands (Fig. 13). An elegant computerized technique to analyse the position of
DMB and cellpeaks has been developed by use of a scanning transmission system [33].
Alternatively, refractive index (n) can be measured in each Percoll fraction and be
correlated to density (d); d 5 6.1905 3 n 2 7.252. A simple type of pycnometry,
weighing of empty and lled micropipettes, is also an accurate method to measure
density. Furthermore, the use of precalibrated non-aqueous organic density gradients is a
most useful technique [34]. Direct measurement of density using a densitometer (DMA,
Anton Paar GmbH., Graz, Austria) is an accurate technique but requires about 1 ml of
sample, which however can be reused.

3.7. Removal of Percoll after centrifugation

Since Percoll is non-toxic to biological material and does not signi cantly adhere to
membranes, it is usually unnecessary to remove Percoll from the puri ed preparations.
After dilution, the cell fractions can be transferred directly to cell culture systems
including serum [35]. In the absence of serum, cells will not attach as it is a well-known
fact that PVP reduces cell adherence to plastic surfaces [36].
It is interesting to note that the infectivity of puri ed viruses in the presence of Percoll
increases compared with controls [15]. One explanation could be that the viruses do not
aggregate in Percoll to the same extent as they do in tissue culture medium.
If it is desirable to eliminate the gradient material, e.g. for electron microscopy,
chromatography by gel ltration on Sephacryl S-1000 (Amersham Pharmacia Biotech,
Uppsala, Sweden) will separate Percoll from larger particles, which are eluted in the
void volume [37]. The Percoll fraction can also be underlaid with e.g. a 50% sucrose
solution and centrifuged for 1 h at 90,000 3 g to pellet the Percoll particles [24].
Recently it was shown that Percoll particles can be removed by mixing Percoll fractions
16 H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30

Fig. 13. Banding of Density Marker Beads (DMB) in a Percoll gradient. Washed parathyroid cells (24 3 10 6 in
1 ml PBS) were layered on top of 8 ml of 40% (v / v) Percoll in PBS in one tube. In a second tube a mixture of
DMB was layered in 1 ml on top of the Percoll solution. After centrifugation for 15 min at 20,000 3 g in
Beckman rotor 40, the distances from the meniscus of the gradient to each bead was measured and compared
to the distances of each visible band of cells in the second tube.

with iodixanol (Optiprep from Nycomed Pharma As, Oslo, Norway) of high density
[38]. After centrifugation at 70,000 3 g for 30 min in a swing-out rotor, organelles will
oat and Percoll start to sediment, and isolated material can be recovered at the
interface.

4. Common applications of cell separation

Both rate zonal and isopycnic separation in Percoll have been used to fractionate the
various cells in blood of human origin and other species as well [32]. Various liver cells,
including hepatocytes, Kupffer cells, liver endothelial cells, stellate cells and bile duct
epithelial cells, from rat have been isolated from a single liver [39]. Leydig cells from
H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30 17

human, mouse, porcine and rat testis have been puri ed to more than 90% in Percoll
gradients [32]. Macrophages from various tissues of human, mouse or rat origin are the
most common cells to be puri ed in Percoll. Mast cells and thymocytes from rat or
mouse have been separated into subpopulations [32]. Cells from muscle, pancreas,
parathyroid glands, placenta and various tumors are often isolated by density gradient
centrifugation. For a general procedure to purify cells in Percoll, see Appendix A. Only
two of the most cited cell applications will be described, see Sections 4.1 and 4.2.

4.1. Sperm preparation for in vitro and in vivo use

4.1.1. Standard protocol to purify sperm cells


Numerous Percoll-based gradients, both continuous and step gradients have been
described for isolation of viable and pure sperm cells [40]. By appropiate dilution with
buffered sperm washing medium, step gradients at 40% and 80% of Percoll are prepared
as described above. The procedure involve rst transfer of 12 ml of the most dense
Percoll solution into the bottom of a sterile centrifuge tube. Secondly 12 ml of the less
dense solution is layered over the rst. Finally 12 ml of liqui ed semen is layered on
top (Fig. 14). After centrifugation at 1500 rpm (400 3 g at 15 cm radius) for 20 min, a
pellet of viable sperm will form at the bottom of the centrifuge tube. If frozen sperm is
used, glycerol, seminal uid and bacteria will remain in the original sample layer while
contaminating cells and non-viable sperm cells band at the interface of 40 / 80% Percoll
and only viable, motile sperm cells penetrate the 80% layer of Percoll. The pellet is
resuspended in 23 ml of an appropriate sperm washing medium and centrifuged at
1200 rpm (250 3 g at 15 cm radius) for 10 min. The sperm pellet is now ready for
analysis or oocyte fertilization.

Fig. 14. Separation of spermatozoa on a discontinuous Percoll gradient. After 20 min of centrifugation the
viable sperms have sedimented to the bottom, while deformed and dead sperms, bacteria and other cells are left
at the 40 / 80% Percoll interface.
18 H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30

4.1.2. Improved sperm cell separation


The sperm cell preparations prepared by Percoll centrifugation are free of mi-
crobiological contaminants and other cells present in the original semen and thus there is
no need for antibiotics in the further sterile manipulations [41]. Production of reactive
oxygen species in sperm, which is negatively correlated to both outcome of the
sperm-oocyte fusion and in vivo fertility, is reduced and there is less reactive oxygen
damage as well [42]. However, the activity of superoxide dismutase (SOD) has also
been lowered in samples prepared by Percoll puri cation [43] and it has been
recommended to add antioxidants to reduce oxygen radical formation and recover a
higher yield of motile sperm [44]. The addition of 10 mM vitamin E acts as an effective,
non-disruptive antioxidant, during the separation and enhances the capacity of human
sperm for sperm-oocyte fusion [42]. Furthermore, it has been shown that electrolyte-free
Percoll gradients containing 0.33 M glucose-0.5% bovine serum albumin preserve the
morphology, motility, viability, and ATP concentration of sperm cells substantially
better than Percoll in NaCl or KCl solutions [45].
It has been suggested that Percoll centrifugation modi es the glycosylation pattern of
structures important for egg fertilization [46]. Percoll could either trigger the early steps
of the acrosomal reaction, thereby inducing an increased expression of sugar moieties on
the sperm cell surface, or select a subpopulation of highly glycosylated sperm. Similarly
the addition of 0.25 mg / ml hyaluronan to thawed sperm cells maintains sperm motility
after puri cation on 40 / 80% Percoll [47]. The addition of 2 mg / ml progesteron to 100%
Percoll causes a signi cant improvement of motility in the puri ed spermatozoa [48].
The improvement in fertilization rate is therefore probably due to other factors besides
simply washing of spermatozoa.

4.1.3. Separation of X-and Y-bearing sperm cells


Although Percoll gradients have been reported to separate X-and Y-chromosome
bearing sperm cells, such gradients have been much more sophisticated [49]. The
separation is based on rate zonal centrifugation rather than isopycnic centrifugation in a
12 step Percoll density gradient. A 94% enrichment of X-chromosome bearing sperm
cells was used by six females and each delivered a female baby without abnormality
[50]. It is also possible to underlayer a X-ray contrasting solution with density 1.15 g / ml
below the Percoll gradient to obtain a higher density at the bottom of the tube. Such
gradients with a density higher than 100% Percoll give a signi cantly increased
separation of X and Y-bearing sperm cells [51].

4.2. Stem cell isolation

Immunotherapy treatment of cancer and of chronic infectious diseases is a new


technology that has been developed during the last decade. It started with the reported
use of isopycnic centrifugation in Percoll in the concentration of human stem cells for
bone marrow transplantation [52]. The majority of hematopoietic precursor cells from
bone marrow are enriched with minimal contamination by other cells at the 40 / 60%
Percoll interface. The procedure takes 3 h and the pH, density and osmolarity can be
H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30 19

controlled. Further puri cation could be achieved by counter ow centrifugation and


depletion of T cells by E-rosetting. Later it was found that it was advantageous to use
peripheral blood progenitor cells in allogeneic transplantation because of the large
number of CD34 cells found in blood. Low density cells banding between 40 and 50%
Percoll contained 47% CD34 1 cells and were depleted of T cells [53]. This enriched
cell fraction can be further treated with monoclonal antibodies and complement to
remove tumor cells. Progenitor cells from human umbilical cord blood have signi cant
advantages over bone marrow cells in terms of proliferative capacity and immunological
reactivity. They are considered as an attractive source of hematopoietic stem cells for
both research and clinical applications. Colony forming cells (CFU) and CD34 1 cells
are highly enriched in gelatin-treated umbilical cord blood and can be further puri ed by
Percoll density centrifugation [54]. A pre-enrichment of CD34 / 45 1 cells may also be
obtained on a 60% Percoll cushion before immunomagnetic separation or processing on
a CD34 1 af nity column [55].
Transfusion of bone marrow cells, splenocytes or blood have enhanced allograft
survival. Co-cultured cells from bone marrow or splenocytes, isolated by Percoll
gradient separation, provide a therapeutic option in the use of cell infusion-based
therapies [56]. Overall, peripheral blood cells, a mixture of T cell blasts, B cells,
dendritic cells, NK cells and immune lineage precursors, isolated in Percoll seem to be
as effective as bone marrow progenitor cells as a source of hematopoietic stem cells for
transplantation and to give an immune response against infected tissue and cancer cells.
One important detail in this procedure to isolate cells is to use a simple reproducible
decanting system by use of a cell trap tube (Dendreon Co., Mountain View, CA, USA).

5. Subcellular fractionation

5.1. The advantages of Percoll in subcellular fractionation

Since subcellular particles are osmotically fragile, Percoll gradients are well suited to
their separation and puri cation. Percoll in itself has a stabilizing effect on organelle
membranes giving high recoveries of enzyme activities [57]. A combination of
isoelectric point and marker enzyme analyses, respectively, has shown that Percoll
density gradients are useful to describe heterogeneity of cell organelles [58]. The system
permitted studies which could not be adequately performed by other techniques. Both
metabolic and catabolic studies could be performed on organelles isolated in iso-osmotic
Percoll gradients. For example a transport mechanism in the catabolism of glycoproteins
from low density to high density lysosomes was found, which later has been con rmed
by other authors in studies on the phagocytic degradation process [59]. Also, unique
phagophores (autophagosomes) have been isolated in a Percoll gradient [24]. Neither
differential centrifugation nor sucrose or Nycodenz (Nycomed Pharma AS, Oslo,
Norway) gradients could separate cytomembranes from autophagosomes. Most probably
the low molecular weight gradient forming components penetrate or shrink the
20 H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30

organelles so that all the subcellular particles attain the same density. They can only be
separated if an osmotically inactive substance such as Percoll is used.
Similar experiences have been reported elsewhere, e.g. in the puri cation of
hydrogenosomes, a key organelle in the metabolism of trichomonads for which sucrose
gradients cannot be used [60]. It is interesting to note that in the cited report Percoll was
centrifuged in a swing-out rotor. The very shallow gradient formed in this rotor seemed
to be effective in separating the various organelles. This is in contrast to our own
experience (see Section 3.3.2).
Although plant mitochondria survive the osmotic damage caused by high sucrose
concentrations in gradients, dilution to iso-osmolar conditions must proceed very slowly
to avoid membrane expansion [61]. However, in Percoll self-generated gradients 95% of
the mitochodria are intact with a markedly higher rate of O 2 uptake than unpuri ed
mitochondria. Furthermore, the massive contamination with microbodies in non-puri ed
mitochondial fractions together with contaminating hydrolases could be eliminated by
the Percoll centrifugation. It is possible that the PVP coat on Percoll acts by binding the
hydrolases.
Note that the isopycnic densities of organelles fractionated in an iso-osmotic Percoll
gradient are markedly different from those observed in hyperosmotic sucrose or salt
gradients [7].
For a general procedure to purify organelles, an example on bacteria isolation from
yogurt in Percoll is shown in Appendix B.

5.2. Enhanced separation of cell organelles

Various techniques have been used to manipulate the density of organelles and thereby
enhance the resolution in Percoll centrifugations.
In order to shift the density of organelles, detergents have been used to lower the
density of lysosomes [62]. Iron-complex has been used to increase the density of
lysosomes [63]. Endocytosis of colloidal gold followed by subcellular fractionation in
Percoll gradients has revealed subpopulations of lysosomes [64].
Salts of appropriate concentrations can have a signi cant effect on the banding of
organelles either by altering the shape of the gradient or by changing organelle volume
and thus the relative density [65]. By substitution of sucrose with salts, to which
mitochondria but not zymogen granules are permeable, the densities of mitochondria are
altered to give a signi cant separation of the two organelles. The incorporation of 100
mM sodium succinate in a Percoll gradient can produce a 70% reduction in mito-
chondrial contamination of zymogen granules.
The addition of 10 mM potassium phosphate accelerates the sedimentation rate of
mitochondria without altering that of lysosomes, resulting in a decreased mitochondrial
contamination of the nal lysosomal fraction prepared in 45% isotonic Percoll [66].
Mitochondria can also be swollen in 2 mM Ca 21 which yields pure lysosomes by
Percoll density gradient centrifugation [67]. It is important to control pH to be 7.4 in
these procedures to avoid contamination by intracellular membranes and plasma
membranes.
H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30 21

6. Some special applications

6.1. Cell synchronization

In 1972 we found that the main population of HeLa cells had densities of 1.0541.058
g / ml in Percoll gradients [68]. When the mitotic index was determined, a sharp peak of
cells in mitosis was found at 1.046 g / ml. Mitotic malignant cells from pleural effusions
[69] and mitotic broblasts are similarly enriched at low density [70]. The mitotic cells
isolated in this way subsequently grow in synchrony in cell culture.
Even though a classic cell cycle does not exist in Plasmodium gametocytogenesis, it
has been possible to enrich fractions of almost all the stages of these gametocytes from
in vitro cultures by use of an improved discontinuous Percoll gradient [71]. In the
mature stage, the gametocytes from the gradient transformed into gametes.
For synchronization of bacteria (Eschericia coli B and K-12, respectively, and
9
Bacillus subtilis) from exponentially growing cultures, about 10 cells was fractionated
by sedimentation in a preformed gradient of 0100% Percoll in growth medium and
centrifuged for only 15 min at 1200 rpm (200 3 g) in a swing-out rotor. The most dense
fraction contained very young, newly divided cells [72]. The newly divided cells
continued to divide synchronously for several generations. Most probably this technique
with short centrifugation time and low g-force separates the cells according to size rather
than density. Woldringh et al. found only a small increase in density at the beginning of
the cycle of bacteria and concluded that the density remains constant and one cannot
synchronize bacteria by density separation [73].

6.2. Unit gravity sedimentation

Unit gravity sedimentation is a common technique to remove cell debris and enzymes
from isolated pancreatic islets by sedimentation through a cushion of 30% Percoll [74].
Very pure islets sediment through the medium and settle on the tube bottom within 10
min. Up to 35 human islets per gram of pancrease can be obtained free from exocrine
tissue by this technique.
In contrast to results with other gradient media both cell debris and non-viable cells
band at a low density in Percoll. The reason for this discrepancy is that Percoll does not
penetrate cells or adhere to membranes and therefore gives physiological values of cell
density [32].
Velocity sedimentation at unit gravity is also a suitable technique for separating cells
differering in size and to remove non-viable cells. To reduce separation time and

increase cell separation capacity, the separation chamber (e.g. Celsep from Sorvall,
Wilmington, Delaware, USA) has to be tilted from a vertical position to a horizontal
position. Up to 10 9 lymphoid cells can be recovered on a discontinuous Percoll gradient
after sedimentation for 3 h at 48C [75]. The larger mast cells from murine peritoneal cell
suspensions can be recovered in 90 min at 48C with a yield of 77% and . 95% purity
[76]. The technique is similar to that described in Fig. 6.
Low Percoll temperature, high cell-suspension density, and the presence of serum in
the media decrease spontaneous release of substances from puri ed cells [77].
22 H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30

6.3. Removal /enrichment of microorganisms

6.3.1. Removal of microorganisms


It is a common practice to employ chemical agents to control microbial infections in,
e.g. tissue cultures. Sedimentation through a 40% Percoll solution is a highly successful
reagent for the recovery of cells free from microorganisms. Cells may be plated directly
without washing and without use of antibiotics or fungicidal agents [78]. It has been
shown that Crytosporidium parvum infected oocytes can be recovered from rat feces free
of most bacteria after centrifugation at low speed, 1700 3 g for 30 min [79]. Further
puri cation of the oocytes can be achieved by isopycnic centrifugation at 16,000 3 g for
20 min on 50% Percoll.

6.3.2. Enrichment of microorganisms


Both bacteria and protozoa from varies species give a high yield and purity after
puri cation on continuous or step gradients of Percoll [32]. Of special interest is the fact
that enrichment media, which interfere with PCR detection, are removed from the
microorganisms and therefore very low detection levels by PCR have been reported after
centrifugation [80].
The ionic strength and osmolarity of Percoll make it very useful for purifying fragile
structures, and the Percoll method allows the recovery of more infectious virus particles
than could be obtained by sucrose centrifugation. That sucrose-CsCl gradients may
degrade a virus can be deduced from the fact that the S-value for the virus decreases in
these low molecular gradient media compared to S-values obtained in Percoll [23].
Percoll does not interfere in electron microscopie studies, DNA and protein analysis,
polyacrylamide gel electrophoresis, immunoprecipitations or biological assays such as
plaque formation on cultured cells.
Of special interest is as noted above that enveloped viruses can give a higher
infectivity after isolation in Percoll depending on the dispersing effects of the colloid
particles [15]. Infectious bovine rhinotracheitis virus (IBRV) was puri ed about 2000-
fold in relation to 3 H-labeled calf cell proteins in a Percoll related medium [81]. Another
virus in the same group, equine abortion virus (EAV), was puri ed to the same extent
with a yield of 75%. When the virus fraction was rebanded in a sucrose gradient
envelope-material was clearly separated from enveloped virus and de-enveloped
particles; i.e. the envelope is disintegrated into fractions of different densities in sucrose
[82].

6.4. Separation of non-bound ligands from bound ligands

Non-bound ligands can be separated from ligands bound to cells or bacteria on


1020% Percoll cushions [83]. Diluted incubation mixtures (0.5 ml) are layered on top
of 10% Percoll (3 ml) in tubes used for a swing-out rotor. After centrifugation for 15
min at 3500 rpm (1350 3 g at 10 cm radius) bacteria or cells sediment through the
gradient medium, whereas the incubation medium, containing unbound ligands remains
as a layer on top of the Percoll. Activity associated with the pellet can then be
quantitated and background activity, from tubes lacking cells or bacteria, is subtracted to
H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30 23

obtain a correct binding estimate. To minimize unspeci c binding of proteins to the tube
walls in this type of experiment, a convenient technique is to coat the tubes with 0.1%
albumin or 1% PVP, although Percoll itself reduces wall adherence [36]. In an assay of
cell adhesion to a protein-coated culture dish the bouyancy can be utilized to separate
the non-adherent cells from the adherent by letting them oat to the top of a dense
Percoll solution (80% solution) in the culture wells [84]. The adherent cells are further
xed to the plate with a dense Percoll / glutaraldehyde xative and quanti ed. It is
interesting to note that no centrifugation step is necessary to perform this kind of very
sensitive assay.

7. Advantages, disadvantages and future perspective

Electron microscopic studies have shown that Percoll is seldom attached to cell
surfaces and there is no evidence for any receptor-mediated uptake [32]. Anyhow, traces
of particles adhered during Percoll density separation can easily be removed from the
surface of cells by simple washing.
If Percoll on the other hand is injected intravenously into rats, ultrastructural
examination of thin sections of liver after 2 h shows a localization of dense silica
particles in perinuclear vacuoles in Kupffer cells. In the liver sections, extracellular 100
nm helical structures, containing electron dense particles, can also be visualized (data not
shown). Recently, it was also shown that the rat visceral yolk sac is capable of capturing
Percoll [85]. The uptake was a true internalization by uid-phase endocytosis as excess
PVP did not decrease the rate of uptake of Percoll.
Studies of the PVP coat on Percoll have shown it to be rmly attached to the silica
[18]. However, there are indications that PVP may interact with serum proteins. Similar
to PVP, Percoll decreases macrophage adherence to plastic at 378C unless serum is
present [86]. It has been shown that the Percoll surface easily can be radiolabelled with
125
I [87]. The body distribution of such radiolabelled Percoll shows that the liver is the
main target organ, while the uptake in other organs is very low (unpublished).
Furthermore, a high amount of radioactivity is found in the urine, which indicates a
liberation of PVP from the Percoll particles in vivo.
To conclude, the PVP-surface of Percoll can be radiolabelled and Percoll is visible
with the electron microscope. Using there tools uptake of Percoll in cells or organelles
have not been seen after experiments at low temperature. In vivo both PVP and silica
particles are recovered mainly in liver.

7.1. Advantages of Percoll

The use of Percoll in the formation of density gradients offers several advantages. In
sharp contrast to pure colloidal silica solutions, Percoll is stable at varying conditions. It
can be mixed with, e.g. seawater of high salt content to isolate microalgae [88]. Another
advantage is that the bouyant density measured in Percoll is directly related to the
physiological composition of a tissue. In the future, not only parathyroid glands may be
examined [89] but also other tissue pieces. The degree of edema in nervous tissue has
24 H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30

already been determined by this technique [90]. A low-cost apparatus with a com-
puterized microgravimetry system facilitates reproducible and accurate results [91].
The major advantage with Percoll is, however, that one can match the osmolarity of
cells or particles to be separated. Sedimenting particles will rest at that point in the
gradient where both Donnan-osmotic equilibrium and buoyant equilibrium are obtained.
Cell viability will be maintained, mitotic cells can be separated and grown in
synchronous culture, puri ed sperm can be used to fertilize oocytes and unique cell
populations for cell therapies can be puri ed on the very shallow gradients formed.
Similarly, the preparation of cell organelles and viruses is facilitated by the maintenance
of iso-osmotic and non-toxic conditions. Another interesting advantage of Percoll is that
it is the over-all density of the hydrated molecule that determines where it bands in a
Percoll gradient [92]. The steric exclusion phenomenon can be utilized to separate
particles with the same density but of different size. The Percoll particles are excluded
from a volume around, e.g. a virus and leaving a hydrated shell, which is determined by
the surface area of the virus and the Percoll radius. The larger the relative size of the
hydration shell the lower the isopycnic density in Percoll. In separation of cells and
organelles the exclusion effects are too small to be signi cant.
It is probably also a steric exclusion effect that limits the size of the ice crystals when
embryos are frozen in the presence of Percoll. It was found that zona pellucida damage
was reduced when the freezing solution contained Percoll, compared to controls, and a
greater number of embryos developed in vitro to the blastocyte stage [93].
Percoll can easily be mixed with heavy X-ray-contrasting agents to utilize the capacity
of these iodinated compounds to penetrate membranes. Sodium diatrizoate has been
added to Percoll to obtain densities around 1.12 g / ml and used to separate light and
dense erythrocytes from fresh human blood [94]. Golgi-derived transport vesicles for
example are thought to be freely penetrated by contrasting agents and should easily be
fractionated in such mixed gradients [95].

7.2. Disadvantages of Percoll

There have been a few reports that Percoll sometimes has given problems in the
isolation of especially sperm cells. A possible endotoxin contamination of some product
batches has been discussed, although this has not been documented [96].
It has been reported that there is an interaction between Percoll and dead sperm,
which results in quenching the uorescence of chromatin stained with Hoechst 33342
(Calbiochem-Behring Co., La Jolla, CA). This nding has been utilized in ow
cytometry / cell sorting to reduce the uorescence of dead sperm [97]. It may well be that
it is the free PVP, in Percoll, which stabilizes the colloidal system that exerts these
properties [18]. However, if an insuf cently high concentration of PVP is present in the
solution, e.g. after washing of cells and the colloid concentration is below 0.1%, one can
observe an aggregation of Percoll in 0.15 M NaCl [17]. One drawback with Percoll is
that it is not possible to autoclave it in the presence of salt [15]. Also, the particles are
too large to be sterile ltered. Sterilization with gamma radiation induces aggregation. In
practice, Percoll has to be autoclaved separately from the saline solution followed by
mixing of the two compounds in a sterile environment.
H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30 25

7.3. Silanized colloidal silica

To overcome the described drawbacks with Percoll, silanized colloidal silica solutions
have been prepared [96] for use e.g in separation of sperm cells of bovine [98] or human
origin [99]. These gradient media can be autoclaved in the presence of saline and have a
low endotoxin content ( , 1 EU / ml). Analysis by liquid chromatography, density
gradient generation and electron microscopy showed no differences between Percoll and
silanized particles (unpublished results).
The silanized colloidal silica particles have been useful to remove contaminants in cell
and microorganism preparations (BactXtractorE, QRAB, Balsta, Sweden; e-mail:
bnor@qrab.se).The ready made solution can be used to purify microorganisms within 1
min centrifugation at 16,000 3 g (14,000 rpm at 7 cm radius) from e.g. food and faeces.
Escherichia O157:H7 was isolated from beef, milk, shrimps, and blue cheese and
allowed 0.5 cfu g 21 to be detected by PCR technique [80].
Already to date, silane silica is used to isolate cell populations that are re-introduced
into the blood stream for immune and hematopoeitic cell therapies [100]. The
formulation of the silanized colloidal silica follows Good Manufacturing Procedures
(GMP) and does not induce a pyrogenic effect, nor does it result in acute toxicity when
injected intravenously as de ned by the United States Pharmacopoeia. Early during this
century it is expected that applications for hematopoietic progenitor cell isolation in the
treatment of viral infections, cancer, etc. will be commercialized [101].

Appendix A. General procedure to separate cells by isopycnic centrifugation

Mix 90 ml Percoll with 10 ml ten times concentrated phosphate buffered saline


(10 3 PBS). This mixture is called 100% Percoll. Layer 10 ml of this solution in the
bottom of a 50 ml Falcon tube.
Dilute 100% Percoll with phosphate buffered saline (PBS) to 50% and 25%.
Tilt the tube to a 458 angle and place a 10 ml pipette in the mouth of the tube and
slowly release rst 10 ml 50% solution, followed by 10 ml 25% solution.
6
Finally add 12 ml of cell suspension ( , 100 3 10 cells) on top of the gradient.
Layer density marker beads (DMB) in a second tube containing the same Percoll
solutions as described above.
Put the two tubes with the same weight in a swing-out rotor and centrifuge for 1015
min at 1500 rpm (400 3 g at 15 cm radius).
After centrifugation, the gradient is displaced upwards through an unloading cap. Use
a stopper with two holes (12 mm diameter), with one connection to the center of the
stopper to collect fractions and with one connection to the bottom of the centrifuge tube
for the introduction of 100% Percoll by use of a peristaltic pump.
Record the density of each fraction (5 ml) and count number of cells in each fraction
by use of phase contrast microscopy. Make a graph of cell distribution versus density. If
desirable washing with PBS (5 volumes to 1 volume of cell suspension) and pelleting of
cells for 10 min at 1500 rpm can eliminate the gradient material.
26 H. Pertoft / J. Biochem. Biophys. Methods 44 (2000) 1 30

Appendix B. Procedure to isolate bacteria from yoghurt

Pipette 8 ml of 40% Percoll in 0.15 M NaCl into each of two ultracentrifuge tubes (10
ml tubes for Beckman angle rotor 40).
To the rst tube add 1 ml of diluted natural yoghurt (1 part yoghurt 1 3 parts of 0.9%
NaCl).To the second tube (reference) tube add instead 1 ml 0.9% NaCl and 0.1 ml
DMB.
Balance the rst tube with cap attached against the second tube 1 cap by adding 40%
Percoll dropwise, using a Pasteur pipette, until the difference is , 0.1 g.
Put the tubes into the rotor, opposite one another. Centrifuge for 15 min at 30,000 3 g
(21,000 rpm at 6 cm radius) at room temperature.
Measure the distance from the meniscus to each colored band in the reference tube
and make a plot of the density pro le. Below the thick white band in the yoghurt tube it
should be possible to discern opalescent bands of bacteria. Record the distances to these
bands and register on the density curve the densities of the various bacteria.
Identify the components by phase contrast microscopy before further analysis.

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