Cytiva Cell Separation Media

Methodology and applications
Cell separation
media
Contents
Introduction 3 How to fractionate and analyze gradients of Percoll 22 Applications 31
Principles of density gradient centrifugation 5 Density determination using Density Marker Beads 22 Blood cells 31
Density Marker Bead — properties 22
Separation by density (isopycnic centrifugation) 6 Effects of ionic strength and sucrose concentration 23 Applications — blood cells 32
Separation by size (rate zonal centrifugation) 6 on density of Density Marker Beads
Using Density Marker Beads 23 Applications — other cell types 41
Percoll – physical properties 7 Other methods for measuring density 24
Fractionation of gradients 24 Applications — microorganisms 51
Particle size composition 7 Cell sorting and counting 25
Viscosity 8 Protein determination and enzyme assay 25 Applications — subcellular particles 53
Density 8
pH and osmolality 8 Removal of Percoll after centrifugation 26 Appendix
Behavior of the colloid 9 Summary methodology charts 61
Washing 26
How to make and use gradients of Percoll 10 High speed centrifugation 26 Scheme 1. Separation of cells on gradients of Percoll 61
Other methods 27 Scheme 2. Separation of subcellular particles on 61
Making and diluting a stock solution of Percoll 10 gradients of Percoll
Diluting stock solutions to lower densities 11 Practical notes 28
The one-step procedure for diluting Percoll 12 References 62
Diluting Percoll to a desired osmolality 13 Care and cleaning of equipment 28
Effects of osmolality on apparent buoyant density of 16 Storage of Percoll 28 Ordering information 76
cells and subcellular particles Sterilization of Percoll solutions 28
Factors affecting gradient formation and shape 17 Aggregates of silica particles 28 Products for cell separation and culture 76
Discontinuous gradients 19 Products for purification of RNA 76
Continuous linear and non-linear gradients 19 Percoll PLUS – low endotoxin 29 Kits for cDNA synthesis 76
Preformed self-generated gradients 20
Gradients formed in situ 20 Physical properties 29
Maximum sample loading 21 Composition 29
A model experiment to standardize conditions 21 Osmolality 29
Density 29 Principles and methodology
Endotoxin activity
Gradients
30
30
handbooks from Cytiva
Practical notes 30 Cytiva offers a wide range of handbooks that provides practical tips
and in-depth information about common methodologies used in
the lab. Visit cytiva.com/handbooks to view the complete list and
download your copies today. 2
Introduction
Since its introduction in 1977, the silica colloid Percoll™ has become the density gradient medium of choice for thousands
of researchers worldwide. Its nearly ideal physical characteristics facilitate its use in separating cells, organelles, viruses,
and other subcellular particles. Percoll is especially useful as a first step to enrich for cell populations before attempting finer
resolution or extraction of nucleic acids. A considerable savings of time and resources may be realized using Percoll as a first
step before employing these methods.
For biological particles, the ideal gradient medium has been described as one having the following characteristics (79):
covers a sufficient density range for isopycnic (Fig 1) banding of all biological particles of interest
possesses physiological ionic strength and pH
is iso-osmotic throughout the gradient
has low viscosity
is non-toxic
will not penetrate biological membranes
will form self-generated gradients by centrifugation at moderate g-forces
is compatible with biological materials
is easily removed from purified materials
does not affect assay procedures
will not quench radioactive assays
3
Percoll is exceptional among the available media in that it fulfills the above criteria, and also provides these additional
advantages:
It can form both continuous and discontinuous gradients
Stability of gradients means that gradients can be premade to give reproducible results
Analysis of gradients is simple with colored Density Marker Beads (available from Cospheric LLC, USA)
Further experiments with isolated materials are not affected by Percoll
The success of thousands of researchers has been documented in the Percoll Reference List
This re-issued manual provides the basic methodology for making and using gradients of Percoll and includes
information on Percoll PLUS, a new silica based colloidal medium optimized for cell separation in clinical research
applications. In addition, the Application Tables in the latter part of this manual provide numerous references for using
Percoll to isolate various cells, microorganisms, organelles and subcellular particles. All experiments described in the
literature using Percoll can also be performed with Percoll PLUS.
4
Principles of density gradient
centrifugation
When a suspension of particles is centrifuged, the sedimentation rate of the particles is proportional to the force applied.
The physical properties of the solution will also affect the sedimentation rate. At a fixed centrifugal force and liquid
viscosity, the sedimentation rate is proportional to the size of the particle and the difference between its density and
the density of the surrounding medium.
The equation for the sedimentation of a sphere in a centrifugal field is:
d2 (rp - rl)
v= ×g
18h
where v = sedimentation rate
d = diameter of the particle (hydrodynamically equivalent sphere)
rp = particle density
rl = liquid density
h = viscosity of the medium
g = centrifugal force
From this equation, the following relationships can be observed:
The sedimentation rate of a particle is proportional to its size
The sedimentation rate is proportional to the difference between the density of the particle and that of the
surrounding medium
The sedimentation rate is zero when the density of the particle is equal to the density of the surrounding medium
The sedimentation rate decreases as the viscosity of the medium increases
The sedimentation rate increases as the centrifugal force increases
5
ρ
Separation by density (isopycnic centrifugation)
In this technique, the density range of the gradient medium encompasses all densities of the sample particles. Each
particle will sediment to an equilibrium position in the gradient where the gradient density is equal to the density
of the particle (isopycnic position). Thus, in this type of separation, the particles are separated solely on the basis of
differences in density, irrespective of size.
Figure 1 illustrates the two types of centrifugal separation (see below for rate zonal centrifugation). When using ρ1
Percoll, it is common to separate particles isopycnically rather than on the basis of size differences (but see Figure 19,
page 31, where both techniques are used.).
Note: When considering biological particles, it is important to remember that the osmolality of the medium can ρ2
significantly alter the size and apparent buoyant density of membrane-bound particles. A high external osmolality will
cause membrane-bound particles to shrink while a low osmolality in the medium will cause the particles to swell. Rate zonal cent. Isopycnic cent. Time
Figure 2 shows that particles centrifuged in gradients of Percoll under physiological conditions (280 to Fig 1. Diagrammatic representation of rate zonal and isopycnic centrifugation.
320 mOsm/kg H2O) have much lower apparent buoyant densities than in sucrose or metrizamide (see also r1 = buoyant density of the less dense (blue) particles
r2 = buoyant density of the more dense (red) particles
Table 1, page 16).
(Courtesy of H. Pertoft, reproduced by kind permission).
Separation by size (rate zonal centrifugation) Percoll

Plasma membranes
In this type of separation, the size difference between particles affects the separation along with the density of Ribosomes Platelets
the particles. As can be seen from the above equation, large particles move faster through the gradient than small Herpes virus
Lymphocytes
particles, and the density range is chosen so that the density of the particles is greater than the density of the
Buoyant density g/mL

Lysosomes Granulocytes
1.1
medium at all points during the separation (Fig 1). The run is terminated before the separated zones reach the bottom Mitochondria Erythrocytes
of the tube (or their equilibrium positions). Nuclei
Sucrose
1.2 Plasma membranes
Lysosomes Mitochondria
Microbodies
Herpes virus
Ribosomes Nuclei
1.3
0 1 2 3 4 5 6 7 8 9
10 10 10 10 10 10 10 10 10 10
Svedberg units S
Fig 2. Approximate sedimentation rates and isopycnic banding densities of particles in a rat liver homogenate,
herpes virus and human blood cells in gradients of Percoll (green) compared with sucrose gradients (blue).
Svedberg units = sedimentation coefficient, 1S = 10-13 s. (27, reproduced by kind permission of the authors
and publisher).
6
Percoll – physical properties
Percoll is available from Cytiva.
Composition silica sol with nondialyzable polyvinylpyrrolidone (PVP) coating
Density 1.130 ± 0.005 g/mL
Conductivity 1.0 mS/cm
Osmolality < 25 mOsm/kg H2O
Viscosity 10 ± 5 cP at 20°C
pH 9.0 ± 0.5 at 20°C
Refractive Index 1.3540 ± 0.005 at 20°C
Percoll is non-toxic Fig 3. Electron microscopy of Percoll particles. Negative contrast with 1% uranyl acetate at pH 4.6
(21, reproduced by kind permission of the authors and publisher).
Particle size composition

The physical properties of Percoll have been extensively studied by Laurent et al. (45, 46, 47). Electron microscopic
examination (Fig 3) shows the silica to be in the form of a polydisperse colloid composed of particles from 15 to 30 nm in
size, with a mean particle diameter of 21 to 22 nm. Hydrodynamic measurements (viscometry and sedimentation) give
values of 29 to 30 nm and 35 nm in 0.15 M NaCl and water, respectively, for the mean particle diameter, indicating a layer
of hydration on the particles which is more pronounced at low ionic strength.
Chromatography of Percoll on Sepharose™ 4B (22) has demonstrated the presence of only 1 to 2% free PVP. Inclusion
of PEG in the eluant did not result in any loss of PVP from the silica, indicating that the PVP is firmly bound. Calculations
based on the nitrogen content of the colloid indicate that the PVP coating is a monomolecular layer.
7
Viscosity
The viscosity of Percoll is a function of the ionic strength, and is lower in saline solutions at physiological ionic strength
(e.g., 0.15 M NaCl) than in water or in 0.25 M sucrose (22).
This has the effect of making gradient formation in 0.15 M NaCl much faster than in 0.25 M sucrose when solutions are
centrifuged under identical conditions (page 17). Under working conditions, the viscosity of Percoll solutions is 1 to 15 cP,
facilitating extremely rapid banding of particles in gradients of Percoll.
Density
Percoll is supplied as a 23% (w/w) colloidal solution in water having a density of 1.130 ± 0.005 g/mL.
Gradients ranging from 1.0 to 1.3 g/mL are achievable by centrifugation as described elsewhere in this booklet. All
biological particles having sedimentation coefficient values of > 60S can be successfully banded on gradients of Percoll,
and most have buoyant densities of < 1.13 g/mL in Percoll (Fig 2).
pH and osmolality
Percoll has a pH of about 9.0, adjustable to pH 5.5 to 10.0 without any change in properties. If the pH is dropped below
5.5, gelling may occur. Gelling can also be caused by the presence of divalent cations, an effect which is exacerbated
by elevated temperatures.
Percoll has a very low osmolality (< 25 mOsm/kg H2O) and can therefore form a density gradient without producing
any significant osmolality gradient itself. This makes it possible to work with density gradients which are iso-osmotic
and adjusted to physiological conditions throughout. This is very important for obtaining preparations of cells having
extremely high viabilities (23), and intact morphology (31). Due to this fact, gradients of Percoll also provide an
opportunity to observe the effect of osmolality on the apparent buoyant density of cells and subcellular particles
(see page 16 and ref. 27).
8
1.14 4 3 2 1
Behavior of the colloid
Percoll particles have an inner core of silica which is very dense (r = 2.2 g/mL) and an average hydrated particle size of 1.12
29 to 30 nm in 0.15 M NaCl and 35 nm in water (46). Thus, when a solution of Percoll (in 0.15 M saline or 0.25 M sucrose)
is centrifuged at > 10 000 × g in an angle-head rotor, the coated silica particles will begin to sediment. This results in an 1.10
Density g/mL
uneven distribution of particles, and thus forms a density gradient. Since Percoll is a polydisperse colloid, its component
particles will sediment at different rates, creating a very smooth gradient. Electron microscopic analysis of gradients 1.08 Starting density
by high speed centrifugation in an anglehead rotor shows that the material at the bottom of the tube is considerably in
enriched in larger particles (Pertoft, personal communication). The gradient forms isometrically (i.e., less dense on top 15 m in
1.06
30 m
and more dense on the bottom) around the starting density and becomes on average progressively steeper with time min m in
0
(Fig 4). Prolonged centrifugation of Percoll at high g-forces results in all the colloid sedimenting to form a hard pellet 6 90
1.04
(see Removal of Percoll, page 26). It is important to note that if a gradient of Percoll is spun at > 10 000 × g in a swinging- 1
bucket type rotor, the colloid will rapidly sediment into a pellet and not form a suitable gradient. 1.02
The colloid does not perceptibly diffuse over time, resulting in the formation of very stable gradients. Therefore, both 2 3 4
discontinuous and continuous gradients can be prepared weeks in advance, giving great reproducibility over the course
0 10 20 30 40 50 60
of an experiment. Distance from meniscus mm
Fig 4. Isometric gradient formation by Percoll in an angle-head rotor, 8 × 14 mL (MSE Superspeed centrifuge)
starting density 1.07 g/mL in 0.15 M NaCl. Running conditions: 20 000 × g for 15, 30, 60 and 90 min. Gradient
density was monitored by means of colored Density Marker Beads (Fig 12) (work from Cytiva Bio-Sciences AB,
Uppsala, Sweden).
9
How to make and use gradients
of Percoll
Making and diluting a stock solution of Percoll
In order to use Percoll to prepare a gradient, the osmolality of Percoll (undiluted) must first be adjusted with saline or cell
culture medium to make Percoll isotonic with physiological salt solutions. Adding 9 parts (v/v) of Percoll to 1 part (v/v) of
1.5 M NaCl or 10× concentrated cell culture medium is a simple way of preparing a Stock Isotonic Percoll (SIP) solution.
Final adjustment to the required osmolality can be carried out by adding salts or distilled water. Cell density depends on
osmolality (Fig 5); because of this, the osmolality of the stock solution should be checked routinely with an osmometer to
ensure reproducibility between experiments. For subcellular particles which aggregate in the presence of salts, the Stock
Isotonic Percoll (SIP) can be made by adding 9 parts (v/v) of Percoll to 1 part (v/v) of 2.5 M sucrose.
The density of the SIP solution can be calculated from the following formula:
(ro- ri) Voro + Vxr10

Vx = Vo thus ri =
(ri - r10) Vx + Vo
where Vx = volume of diluting medium (mL)
Vo = volume of undiluted Percoll (mL)
ro = density of Percoll (1.130 + 0.005 g/mL*)
r10 = density of 1.5 M NaCl = 1.058 g/mL (minor differences for other salts)
density of 2.5 M sucrose = 1.316 g/mL (minor differences for other additives)
ri = density of SIP solution produced (g/mL)
Thus, for SIP in saline, ri = 1.123 g/mL and for SIP in sucrose, ri = 1.149 g/mL, assuming ro = 1.130 g/mL.
* Exact density as stated on the Certificate of Analysis, which can be found under Literature at www.cytiva.com.
10
Diluting stock solutions to lower densities
ro = 1.135
Density g/ml
ro = 1.130
ro = 1.125
1.14
Solutions of Stock Isotonic Percoll (SIP) are diluted to lower densities simply by adding 0.15 M NaCl (or normal strength
ro = 1.135
cell culture medium) for cell work, or with 0.25 M sucrose when working with subcellular particles or viruses. 1.12 ro = 1.130
ro = 1.125
The following formula can be used to calculate the volumes required to obtain a solution of the desired density.
1.10
se
(ri - r)
o
cr
Su
M
Vy = Vi
5
1.08
0.2
th
(r - ry)
wi
ion
Cl
Na
lut
1.06
Di
5 M
0.1
where Vy = volume of diluting medium in mL
th
wi
ion
1.04
lut
Vi = volume of SIP in mL
Di
ri = density of SIP in g/mL 1.02
ry = density of diluting medium in g/mL

(density of 0.15 M NaCl is ~1.0046 g/mL) * 10 20 30 40 50 60 70 80 90
% of Stock Isotonic Percoll (SIP) in final solution
100
(density of 0.25 M sucrose is ~1.032 g/mL)*

Fig 5. Dilution of Stock Isotonic Percoll (SIP) with iso-osmotic saline or sucrose solution. Po is the density of
r = density of diluted solution produced in g/mL the Percoll (undiluted). SIP is prepared as described on page 12. The calibration lines shown are for guidance
only. For accurate density measurements, refer to the formula given in the text (work from Cytiva Bio-Sciences
Example: To dilute 55 mL of SIP to a final density of 1.07 g/mL, determine the amount of 0.15 M NaCl required. AB, Uppsala, Sweden).
1.123 - 1.07 Note: The graph shown in Figure 5 can also be used as an empirical guide to the
Volume of 0.15 M NaCl required = 55 × density of solutions produced by diluting SIP with 0.15 M saline or 0.25 M sucrose.
1.07 - 1.0046
This graph refers to the dilution of SIP where SIP is 90% (v/v) undiluted Percoll
= 44.6 mL osmotically adjusted by addition of 10% (v/v) saline or sucrose. To avoid confusion,
it is therefore preferable to refer to the actual density of the working solution (or to
The above formula is useful for achieving densities that will be very close to the actual density desired. However, slight
state % SIP) rather than to refer to the solution as a percentage of Percoll in
variations in volumes and densities of diluting media will affect final density. For determining actual densities, we
iso-osmotic saline or sucrose. This is particularly important when using the one-step
recommend measuring the final density of Percoll solutions using a densitometer or refractometer (see page 24).
dilution procedure described below, where a working solution of known density is
obtained by diluting Percoll (undiluted) plus concentrated salts or sucrose to a final
volume with distilled water.
* from CRC Handbook of Chemistry and Physics, 67th edition (1986-1987), CRC Press, D253 and D262.
11
The one-step procedure for diluting Percoll
Percoll (undiluted) may de diluted directly to make a final working solution of known density by the following procedure. Example: To prepare 100 mL of working solution of Percoll of density 1.07 g/mL in
In a measuring cylinder, add 1.5 M NaCl or 2.5 M sucrose to 1/10 of the final desired volume (e.g., 10 mL for 100 mL of 0.15 M NaCl. To 10 mL of 1.5 M NaCl, add
working solution). To this, add the required volume of Percoll (undiluted), calculated using the formula shown below.
Make up to the final volume with distilled water. 1.07 - 0.1058 - 0.9
Volume of Percoll required = 100 ×
0.13
r - 0.1r10 - 0.9 = 49.4 mL (if Percoll density is 1.130 g/mL) and make up
Vo = V to 100 mL with distilled water.
ro - 1
where Vo = volume of Percoll (undiluted) (mL) The above formula is useful for achieving densities that will be very close to the
actual density desired. However, slight variations in volumes and densities of
V = volume of the final working solution (mL) diluting media will affect final density. For determining highly accurate densities, we
r = desired density of the final solution (g/mL) recommend measuring the final density of Percoll solutions using a densitometer or
refractometer (see page 24).
ro = density of Percoll (undiluted) (g/mL)
(see Certificate of Analysis for exact density) Graphs similar to the one shown in Figure 5 can be drawn to relate the volume of
Percoll (undiluted) to the final density.
r10 = density of 1.5 M NaCl = 1.058 (g/mL)
(minor differences for other salts)
density of 2.5 M sucrose = 1.316 (g/mL)
(minor differences for other additions)
12
Diluting Percoll to a desired osmolality
To make isotonic Percoll for most mammalian cells, it is common to dilute 9 parts of Percoll (undiluted) with 1 part of
1.5 M NaCl or 2.5 M sucrose solution. This Stock Isotonic Percoll (SIP) is then further diluted with physiological buffers
according to needs. However, while this procedure has proved successful, it is rather simplistic and does not take into
account the effect of having solid silica particles present (i.e., that 100 mL of Percoll stock contains a certain volume of
solid silica, making the total aqueous volume less than 100 mL). Due to the volume occupied by silica, the electrolytes
in the stock solution have a higher effective concentration than in physiological salt solution, and SIP made in this way
will be hyperosmolal. Thus, determining the actual osmolality of the SIP has always been recommended.
Vincent and Nadeau (555) discuss the problem elegantly and described an equation which can be used to calculate the
number of parts of Percoll which should be added to one part of 10× concentrated physiological salt buffer to obtain a
SIP of any desired osmolality. The authors determined the fraction of the total volume of a Percoll stock solution which
is occupied by silica and thus determined the ratio of volume of aqueous solution to that of total Percoll stock solution.
Oc - Of
Vp = Vc
R(Of - Op)
where Vp = number of parts of Percoll to be added
Vc = number of parts of solute concentrate (e.g., 1.5 M NaCl) to be added
Oc = osmolality of solute concentrate (e.g., 1.5 M NaCl = 2880 mOsm)
Of = desired osmolality
R = ratio of aqueous volume to total volume of Percoll (typically = 0.85 for NaCl and 0.80 for sucrose)
Op = osmolality of Percoll undiluted (see Certificate of Analysis)
13
The key variable in this equation is R, which is a measure of the real aqueous volume of a Percoll solution. The value of R
is a function of the hydrodynamic volume occupied by the Percoll particles. This, in turn is a function of the ionic strength
of the medium; that is, as ionic strength increases, hydrodynamic volume decreases. Thus, there is a difference in the R
value of 1.5 M NaCl and 2.5 M sucrose.
To obtain a SIP of osmolality = 320 mOsm/kg H2O adjusted with 1.5 M NaCl (i.e., 10× concentrated physiological saline):
2880 - 320
Vp = 1 = 10.04
0.85 (320 - 20)
assuming: 2880 = osmolality of 1.5 M NaCl

(10× concentrated physiological saline)
20 = osmolality of Percoll undiluted
Therefore to obtain a SIP of 320 mOsm/kg H2O, one would add 10 parts Percoll to 1 part 1.5 M NaCl.
The ratio of concentrated solute solution (i.e., 1.5 M NaCl, etc.) to SIP is called Rx where:
Vc
Rx =
Vp + Vc
14
Using this formula, one can calculate the amount of Percoll (undiluted) required to make a final working solution of known
density and osmolality.
r - Rxr10- (1-Rx)
Vo = V
ro - 1
where Vo = Volume of Percoll undiluted (mL)
V = Volume of final working solution (mL)
r = desired density of final working solution (g/mL)
Rx = fraction of total volume which is solute concentrate (i.e., 1.5 M NaCl, etc.)
ro = density of Percoll undiluted (g/mL)

(see Certificate of Analysis)
r10 = density of 1.5 M NaCl (1.058 g/mL), 2.5 M sucrose (1.316 g/mL), etc.
Thus, for 100 mL of SIP of osmolality = 320 mOsm/kg H2O adjusted with NaCl and density = 1.07 g/mL:
1.07 - 1/11 × 1.058 - (1-1/11)

Vo = 100 = 49.8 mL
1.13 - 1
The final solution contains 9.1 mL of 1.5 M NaCl (1/11 × 100 = 9.1), 49.8 mL Percoll undiluted and 41.1 mL
(i.e., 100 - 58.9 = 41.1) of distilled water.
15
Hepatocytes
Effects of osmolality on apparent buoyant density of cells and 1.15
200
(viable) 30
subcellular particles
Number of cells × 106

300 mOsm
Density g/mL
400
1.10 20
The very low osmolality of Percoll has facilitated the study of the interrelation of the separation medium osmolality
with the apparent buoyant density of particles. Figure 6 shows the effects of banding rat liver hepatocytes in
Percoll gradients having osmolalities of 200, 300 and 400 mOsm/kg H2O. The apparent buoyant density of the cells ty gradi
ent
1.05 ensi 10
increases with increasing osmolality, due to removal of water from the cells. The same effect has been observed with D
Non-viable
mitochondria (Fig 7) and with lysosomes (Table 1). Even small changes in osmolality cause a large change in the cells
apparent buoyant densities of these organelles. The actual recorded buoyant densities of particles banded in Percoll
gradients at physiological osmolality are therefore much more likely to correspond to those existing in vivo, than when 1 2 3 4 5 6 7 8
the particles are banded in sucrose or other centrifugation media. Distance from meniscus cm
Fig 6. Fractionation of rat liver hepatocytes cells (35 × 106 cells in a volume of 2 mL) on a self-generated
Table 1. Changes in buoyant density of lysosomes after incubation in serum albumin Percoll gradient (8 mL solution with a density of 1.065 g/mL). The osmolality of the Percoll solution was varied
by adding NaCl to 200 mOsm, 300 mOsm and 400 mOsm. Centrifugation was performed in a Beckman rotor
Incubation medium Osmolality of medium Average density of 30.2 for 15 min at 35 000 × g at a temperature of 4°C. Density gradient determined using Density Marker
Albumin % Sucrose % (mOm/l) lysosomes (g/mL) Beads (see page 22) (27, reproduced by kind permission of the authors and publisher).
- 8.5 284 1.045
2.5 8.5 288 1.058
1.15
5 8.5 292 1.074 70
Enzyme activity % of total

7.5 8.5 300 1.078 60
10 8.5 310 1.091
Density g/mL
50 1.10
20 8.5 374 1.110 40
30 8.5 503 1.148 30

1.05
40 8.5 800 1.177 20
10 en t
Dens ity gradi
A lysosomal fraction from rat hepatocytes was recovered from a Percoll/0.25 M sucrose gradient at a density of 1.0 to
0
1.05 g/mL and incubated in the media described in the table for 1 h at 37°C. The buoyant density was then redetermined 0 5 10 15 20
in a gradient of Percoll/0.25 M sucrose (27, reproduced by kind permission of the authors and publisher). Fraction number
Fig 7. The density distribution of mitochondria from rat liver cells after incubation in iso-osmotic buffer
(orange) and buffer containing 17.5% albumin (green). Centrifugations were performed in a Beckman 65 rotor
(23° angle) for 30 min at 40 000 × g (59, reproduced by kind permission of the authors and publisher).
16
Factors affecting gradient formation and shape
Although the hydrated volume of Percoll particles is smaller in the presence of 0.15 M NaCl than in 2
Percoll/0.25 M sucrose, the sedimentation rate of the particles is faster due to the lower viscosity of Percoll 40º
in saline. Thus, when Percoll is made iso-osmotic with a final concentration of 0.15 M saline or a tissue culture 4
medium of equivalent ionic strength, it will form a self-generated gradient about 2 to 3 times faster than the
6
equivalent Percoll solution made iso-osmotic with a final concentration of 0.25 M sucrose.
Centrifugation and time are interrelated in that it is the total (g-force) × (time) which determines the shape
Distance from the meniscus cm

of the gradient. A minimum of approximately 10 000 × g should be used for Percoll in 0.15 M saline and about 2
25 000 × g for Percoll in 0.25 M sucrose in order to self-generate gradients in anglehead rotors. Rotor geometry 23.5º
has a marked effect on gradient shape under given conditions as shown in Figure 8. As the angle approaches 4
vertical, the pathlength for formation of the gradient becomes shorter and the gradient forms more rapidly.
Figures 9 and 10 demonstrate that the initial concentration of Percoll also has some effect on the shape of 6
the gradient formed.
2
14º
1.00 1.10 1.20 Density g/mL
Fig 8. The effect of rotor angle on gradient development using Percoll. Starting density was 1.065 g/mL in
0.15 M NaCl. Running conditions: 30 000 × g for 14 min. Colored lines refer to the positions of the colored
Density Marker Beads (45, reproduced by kind permission of the authors and the publisher).
17
1.14
Centrifugation in vertical rotors will form gradients of Percoll very rapidly. Care must be taken, however, to ensure that
the compacted pellet of Percoll which may be formed under high speed centrifugation conditions does not contaminate 1.12
the gradient during fractionation. 90%

1.10
The use of swinging bucket rotors for self-generation of gradients is not recommended, due to the long path length and 80%
Density g/mL
unequal g-force along the tube. However Jenkins et al. (personal communication and ref. 87) report some advantages in 1.08
70%
using these types of rotors for subcellular fractionation of liver organelles. 60%
1.06
Zonal rotors can be used to form gradients of Percoll in situ. Gradients formed in zonal rotors have the same 50%
characteristics as those generated in angle-head rotors. Because of their large sample volumes, it is recommended that 1.04
40%
30%
the separation conditions in a nonzonal rotor be empirically determined prior to scale-up in a zonal rotor. Zonal rotors
have been used in the large scale purification of viruses (21) and for subfractionation of lysosomes (24). 1.02
20%
When starting work with self-generated gradients, it is advisable to conduct a model experiment with colored Density 60 50 40 30 20 10
Marker Beads (see page 21) to produce a series of standard curves under known conditions which are characteristic of Band position from bottom of tube mm
the angle-head rotor to be used for subsequent experiments. Fig 9. Use of colored Density Marker Beads to show gradient shape. Gradients formed from solutions of Percoll
varying from 90% to 20% of stock isotonic Percoll in 0.15 M NaCl. Running conditions 23° angle-head rotor
30 000 × g, 15 min (work from Cytiva Bio-Sciences AB, Uppsala, Sweden).
1.14
1.12
1.10
Density g/mL
90%
1.08 80%
70%
1.06 60%
50%
40%
1.04 30%
20%
1.02
60 50 40 30 20 10
Band position from bottom of tube mm
Fig 10. Use of colored Density Marker Beads to show gradient shapes. Dilutions of Percoll as in Figure 9,
running conditions: 23° angle-head rotor, 60 000 × g, 15 min. Steeper gradients were formed by the greater
g‑force (work from Cytiva Bio-Sciences AB, Uppsala, Sweden).
18
Discontinuous (step) gradients Banded cells
Density of Percoll solution g/mL

Discontinuous gradients offer great flexibility and ease of use. Often, only a cushion of Percoll or a single step is all that
1.004
is required to achieve excellent enrichment or resolution of a target cell type. For example, most blood cells can be 7
enriched using discontinuous gradients (66,69) (Fig 11). 1.062
6
Fraction number
To form a discontinuous gradient, SIP is diluted to a series of different densities as described on page 10. The solutions 1.064
of different density are then carefully layered in order of density one on top of another, starting with the most dense 390 × g 5
at the bottom of the tube. This is most conveniently done using a pipette or a syringe fitted with a wide-bore needle.
1.066
30 min 4
It is important to keep the tip of the instrument against the wall of the tube just above the surface of the liquid to avoid 1.068
a ”splash” and mixing at the interface. Formation of a sharp band of cells at a interface will occur only if there is a sharp 3
change in density. 1.070
2
Centrifugation is performed using relatively gentle condition, such as 400 × g for 15 to 20 min in a bench-top centrifuge. 1.080
These gentle conditions result in the isopycnic banding of cells at the relevant interfaces. The low-g conditions and short 1
run time will not cause sedimentation of the Percoll and will not affect the gradient in any way.
PBMC in
Continuous linear and non-linear gradients Percoll solution
Continuous gradients are characterized by a smooth change in density from the top to the bottom of the tube. Instead Fig 11. Separation of lymphocytes and monocytes by discontinuous density centrifugation in Percoll.
of the obvious interfaces present in the discontinuous gradient, a continuous gradient can be thought of as having an 1.5 to 2.0 × 107 PBMC (peripheral blood mononuclear cells) isolated on Ficoll-Paque™ were mixed in 11.25 mL
infinitive number of interfaces. Therefore, isopycnic banding of cells occurs at the precise density of the cell. of Percoll in Hanks BSS containing 1% HEPES buffer (density = 1.080 g/mL) and underlayered below the steps
shown in the figure (69, reproduced by kind permission of the authors and publisher).
To form such a gradient, SIP is first diluted to produce two solutions of known density at the limits of the range required,
and then mixed using a dual-chamber gradient maker (e.g., Cytiva Gradient Mixer GM-1). A linear gradient spanning the
range between the limits of the two starting solutions is formed.
A single-channel peristaltic pump (e.g., Cytiva Peristaltic Pump P-1) in combination with a gradient mixer can be used
to generate linear, convex, and concave gradients, depending upon the relative diameters of the tubing used. A very
narrow range of densities from top to bottom of the gradient can be formed to effect a maximum resolution of viable
cells. Heavier cells usually pellet, while non-viable cells are found at the top of the gradient. For example, erythrocytes
will pellet if the density at the bottom of the gradient does not exceed 1.08 g/mL. Density Marker Beads can be used
as an external marker in a tube containing an identical gradient to that in the sample tube.
The centrifugation conditions necessary to achieve a separation are the same as those for the discontinuous gradients.
Examples of separations performed on continuous gradients include the purification of Leydig cells (31), lactotrophs (19),
bone marrow cells (52), intestinal epithelial cells (18), marine microalgae (28, 60) and chloroplasts (49, 58, 76, 88, 109).
19
Preformed self-generated gradients
Preforming a gradient by centrifugation can be a convenient alternative to using a gradient maker or pump. As described
earlier, Percoll will sediment when subjected to significant g‑forces (i.e., > 10 000 × g). When preforming a gradient, SIP is
diluted to a density that lies in the middle of the range in which maximum resolution is required. Two centrifuge tubes
are filled with gradient material (one for the experiment and one containing Density Marker Beads). This second tube
serves both as a counter-balance and as an external method for monitoring the gradient. The tubes are centrifuged in
an angle-head rotor (e.g., 30 000 × g for 15 min), and the gradient forms isometrically around the starting density (Fig 4).
The relatively ”flat” region of the gradient should encompass the range required for maximum resolution of the target
cells. This can be confirmed by observing the shape of the gradient in the tube containing the Density Marker Beads.
The gradient becomes progressively steeper with time. It has been shown that the shape of the gradient is approximately
linear related to the total g-force and time of the centrifugation (22).
After forming the gradient, isopycnic banding of cells can be accomplished by low-speed centrifugation for 15 to 20 min
at 400 × g. If an estimate of cell density is required, a volume equal to that of the cell suspension is layered on top of the
tube containing the Density Marker Beads. This serves as both a way to estimate cell density and as a counter-balance.
Gradients formed in situ

The sedimentation coefficients of subcellular particles and viruses are usually too low to allow banding on preformed
gradients at low g-forces. Therefore, it is often convenient to mix the suspension of biological particles with Percoll and
to band the particles on a gradient formed in situ. Gradients of Percoll formed by centrifugation are metastable (i.e., they
will change continuously during high speed centrifugation). The rate of sedimentation of the colloid is slow enough to
allow the banding of small viruses and cell organelles with ”S” values > 60S as the gradient is formed in situ.
A common method for forming gradients in situ is to prepare a SIP, using 9 parts of Percoll to 1 part of 2.5 M sucrose.
The SIP is then diluted to the desired density using 0.25 M sucrose. (Although sucrose is typically used to make in situ
gradients, cell culture media can also be used). When mixing the sample directly with gradient material, the effect on the
overall density of the Percoll solution can be calculated from the formula on page 11. Premixing of the sample with the
gradient material is convenient when it is desirable to accurately measure the buoyant density of the particles. However,
it may be better to layer the experimental sample on top of the gradient material, particularly in cases where it is
desirable to separate subcellular particles from soluble proteins. The soluble proteins will remain in the buffer layer above
the gradient and subcellular particles will separate in the Percoll gradient in situ.
Centrifugation must be carried out in an angle-head rotor. A balance tube containing Density Marker Beads in place of
experimental sample is used to monitor the gradient. An appropriate model experiment similar to the one described on
page 21, should be carried out first to establish the gradient formation characteristics of the rotor to be used.
20
Maximum sample loading
The example chosen is for 10 mL gradients, but this may be scaled up for
There are no standard rules governing the maximum quantity of cells or subcellular material which can be separated
larger tube sizes.
on gradients of Percoll. For subcellular fractionation, successful purification can be achieved with a total loading of
1 to 5 mg of protein in a samlpe volume of 0.5 mL on 10 mL of gradient material (Pertoft, personal communication). 1. Mix 49.5 mL of Percoll with 5.5 mL of 1.5 M NaCl to make a SIP.
2. Mix SIP from step 1 with 0.15 M NaCl to make a series of 10 mL
A model experiment to standardize conditions experimental samples (total centrifuge tube size = 13.5 mL) as shown
The exact shape and range of gradients formed during centrifugation is influenced by the model and angle of the rotor in the following table:
used, and by the size of the centrifuge tubes. The following experiment is designed to enable you to establish a series
of gradient curves for a particular rotor and tubes, and can be used as a reference for all future experiments. Tube No. 1 2 3 4 5 6 7 8 9 10
Percoll (SIP) (mL) 10 9 8 7 6 5 4 3 2 1
The experiment can be repeated using Percoll in 0.25 M sucrose; in this case, running conditions should be 50 000 × g
for 25 min followed by 100 000 × g for 25 min. 0.15 M NaCl (mL) - 1 2 3 4 5 6 7 8 9
3. Add 10 µl of a suspension of each type of Density Marker Beads to each

tube according to the instructions supplied in the pack.
4. Balance and cap the tubes, and mix them by inverting several times.
5. Place the tubes in the angle-head rotor (if there are only 8 spaces, omit
tubes 1 and 10).
6. Centrifuge at 30 000 × g for 15 min.
7. Carefully remove the tubes and using millimeter graph paper, measure
to the nearest 0.5 mm the distance of each band from the bottom of the
tube.
8. Plot the gradient shape for each tube by calibrating each band with the exact
buoyant density for each Marker Bead.
9. Re-mix the contents of each tube by inversion and repeat the centrifugation,
this time using 60 000 × g for 15 min.
10. Measure the gradients and plot the results as before. Calculate the exact
density of the dilution using the formula (see page 11). Figures 9 and 10 show
typical examples of a series of curves generated using Percoll in 0.15 M NaCl.
21
How to fractionate and analyze
gradients of Percoll
Density determination using Density Marker Beads
Using Density Marker Beads as an external marker facilitates monitoring of the gradient shape and range. The position
of cells or organelles within the gradient may be accurately located before fractionation using preformed gradients (73, 83).
The densities of the Density Marker Beads cover the buoyant densities of the vast majority of cells and organelles to be
separated in Percoll. In addition to providing a very rapid and simple method for density measurement, using Density
Marker Beads provides more accurate data than other methods, since distortion of gradients by fractionation before
analysis is completely avoided. 15 min
Density Marker Beads are also very useful for standardizing running conditions before carrying out an actual experiment,
using the model experiment described previously to generate a series of gradient curves specific for a particular rotor 30 min
60 min
and tube type. 90 min
Density Marker Bead — properties

Each vial contains freeze-dried cross-linked dextran beads having an accurately determined density in Percoll. Nine
of the ten bead types can be used for gradients of Percoll containing 0.15 M NaCl or 0.25 M sucrose. Vial 5 is used
exclusively for Percoll with 0.15 M NaCl and vial 10 contains beads to be used only for Percoll with 0.25 M sucrose.
Fig 12. Banding of Density Marker Beads in gradients of Percoll as described in Figure 4 (work from Cytiva
Volume of beads swollen in water: 0.7 mL/vial Bio-Sciences AB, Uppsala, Sweden).
Density of each bead type: calibrated to ± 0.0005 g/mL

Total density range covered: 1.017–1.142 g/mL for Percoll in 0.15 M NaCl
1.037–1.136 g/mL for Percoll in 0.25 M sucrose
For detailed information on the properties and use on Density Marker Beads please refer to manufacturer’s technical
information and instructions for use.
22
Effects of ionic strength and sucrose concentration on density of Density Marker Beads 1.16 9 1.16
9
8
The actual buoyant density of the beads will vary slightly with ionic strength or sucrose concentration (osmolality).
Figure 13 shows variations of density with ionic strength and Figure 14 shows variations with sucrose concentration. 8

1.12 1.12 7
When working with systems outside the normal range of ionic strength or osmolality, these figures may be used as a 7 6
guideline for calibration of bead densities. 6 10
1.08 5 1.08 4
Figure 15 shows the correlation of densities calibrated with Density Marker Beads and by a digital densitometer. This latter
4 3
method may be used as a crosscheck when working with Percoll in systems outside normal physiological conditions. 2
3 1
Using Density Marker Beads 1.04 2 1.04
The beads must be swollen with water prior to use; 1.0 mL of sterile water is added to each vial and the beads are allowed 1
to swell overnight. For long term storage of beads in water, it is advisable to add a preservative such as Merthiolate™
1.00 1.00
(0.01% w/v). 0.05 0.15 0.25 0.10 0.20 0.30
Concentration of NaCl (M) Concentration of sucrose (M)
The quantity of beads required for each experiment will depend on the size of the centrifuge tube, but 10 to 15 µl of
suspension is usually sufficient for 10 mL of Percoll. When dispensing the beads with a micropipette, it is useful to snip
Fig 13. Effects of salt concentration on the recorded Fig 14. Effects of sucrose concentration on the
off the end of the disposable plastic tip to avoid clogging by the beads. densities of Density Marker Beads in gradients of recorded densities of Density Marker Beads in
The size of the Density Marker Beads is sufficiently small for them to pass through tubing, monitoring equipment, etc., Percoll. Numbers refer to different bead types (work gradients of Percoll. Numbers refer to different
from Cytiva Bio-Sciences AB, Uppsala, Sweden). bead types (work from Cytiva Bio-Sciences AB,
without problems. Density Marker Beads have been used to monitor gradients of Percoll in zonal centrifuge rotors. Uppsala, Sweden).
Density Marker Beads are used as external markers, in a centrifuge tube containing identical gradient material to the
one used for the experiment. They should not be mixed with the cell sample. Density Marker Beads are added to the
1.15
control tube, which is then used as a counter-balance in the rotor during the centrifugation. The shape of the gradient Density Marker Beads
Digital densitometer
is measured as described in the model experiment on page 21.
Density g/mL
For detailed information on the properties and use on Density Marker Beads please refer to the manufacturer’s technical 1.10
information and instructions for use.
1.05
20 30 40 50 60 70
Volume (mL)
Fig 15. Correlation of recorded densities of a Percoll gradient in 0.15 M NaCl using Density Marker Beads and
a digital densitometer (DMA 46, Anton Paar A.G.). Fraction size 2.64 mL, centrifuge MSE Superspeed 75, rotor
10 × 100 mL, angle 18°, 40 000 × g for 60 min (work from Cytiva Bio-Sciences AB, Uppsala, Sweden).
23
Other methods for measuring density 1.36
Several techniques can be used to monitor the density of Percoll solutions after fractionation. Weighing of empty and filled e
c ros
glass micropipettes is accurate but tedious. It is also possible to measure the isopycnic equilibrium point of samples in a Su
5 M
precalibrated gradient made from nonaqueous organic liquids (12). Refractive index has a linear correlation with the density 0.2
Refractive Index
1.35 with
of a Percoll solution as shown in Figure 16. Direct measurement using a densitometer (e.g., DMA 3, Anton Paar A.G.) is an o ll l
rc C
accurate alternative to using Density Marker Beads (Fig 15). Pe Na
.15
i n 0
o ll
Fractionation of gradients 1.34 Perc
After centrifugation, the gradient can be fractionated by puncturing the bottom of the tube and collecting the outflow
into fractions, or by a number of other techniques (1, 28). A simple and convenient method is to collect the fractions from
the top of the tube by displacement with a dense medium such as undiluted Percoll, or a 60 to 65% sucrose solution. 1.33
Upon pumping this dense material to the bottom of the tube, fractions can be drawn off the top. Zonal rotors may be
emptied by pumping a denser solution to the distal part of the rotor and collecting fractions from the center. 1.00 1.05 1.10 1.15 1.20
Density g/mL
Fig 16. Refractive index as a function of density of a Percoll gradient (work from Cytiva Bio-Sciences AB,
Uppsala, Sweden).
24
Cell sorting and counting
Percoll does not interfere with fluorescent activated cell sorting (FACS) (911, 1042), or with electronic counting
instruments (12). The DNA content of gradient fractions can also be used as a measurement of cell number (12).
Protein determination and enzyme assay

Percoll causes a background color with Folin-Ciocalteau and Lowry reagents, and all measurements should use Percoll
solutions for the preparation of the blank. Higher protein concentrations can be determined using the biuret reaction
(85). Terland et al. (89) recommend the Coomassie™ Blue method of Bradford (90), since Percoll does not interfere
with color development. Vincent and Nadeau (518) have reported a modification of Bradford´s method which involves
precipitation of Percoll in a NaOH Triton™ X-100 mixture.
Cell organelles are often identified primarily by the presence of specific enzymes. Many enzyme assays can be carried out
in the presence of Percoll without interference. Pertoft and Laurent (21) described an experiment in which the enzymes
5’-nucleotidase (plasma membranes), glucose-6-phosphatase (microsomes), b-glucuronidase (lysosomes) and succinic
dehydrogenase (mitochondria) from rat liver homogenates were analyzed in the presence of Percoll. In all cases, the
activities were at least as high in Percoll as in the controls indicating that the determinations were not influenced by
the medium. Labile succinic dehydrogenase activity was stabilized by Percoll. Aryl sulphatase, alkaline phosphatase,
acid phosphatase, b-galactosidase, N-acetyl-a-D-glucosaminidase and b-glucosaminidase have also been analyzed
in the presence of Percoll without interference from the medium (21). Due to light scattering by Percoll, it is preferable
to use enzyme assays which utilize fluorescence rather than absorbance for detection of activity. For further details of
enzyme measurements in Percoll, see references 13, 43, 53, 54, 78 and 89.
25
Removal of Percoll after
centrifugation
Since Percoll is non-toxic to biological materials and does not adhere to membranes, it is usually unnecessary to remove
Percoll from the purified preparation. Cells can be transferred directly to cell culture systems (23, 57), virus infectivity is
unimpaired (21), and organelles can be used for metabolic studies (21) without any effect caused by the gradient material.
The following methods can be used to eliminate the gradient material if desirable.
Washing (low speed centrifugation)

Living cells can be separated from Percoll medium by washing with physiological saline (5 volumes saline to 1 volume of
Percoll cell suspension). The washing may be repeated two or three times and the cell collected between each washing
step by centrifugation at 200 × g for 2 to 10 min. Studies with radioactively labeled Percoll (Table 2) have shown that no
detectable residual Percoll is left adhering to cells washed in this way. Electron micrographs by Enerbäck et al. (9) (Fig 17)
and Schumacher et al. (31) show cell preparations with no visible contaminating particles from the gradient material.
Fig 17. Electron micrograph of mast cells isolated by gradient centrifugation on Percoll (9, reproduced by kind
Washing (high speed centrifugation) permission of the authors and publisher).
For viruses and subcellular particles which are too small to be pelleted by low speed centrifugation as described above,
the biological material can be separated from coated silica particles by high speed centrifugation in a swinging bucket Table 2. Removal of Percoll from rat liver hepatocytes
rotor or angle-head rotor. The undiluted fraction obtained from the first centrifugation run is placed in a centrifuge tube I-labeled Percoll was used to isolate hepatocytes in Eagle's MEM at a
125
and spun in a swinging bucket rotor at 100 000 × g for 2 h, or 90 min in an angle-head rotor (100 000 × g) to pellet the density of 1.07 to 1.09 g/mL.
Percoll. The biological material remains above the hard pellet of Percoll (12, 39). 125
I (cpm)
5 mL of the original cell suspension in Percoll 35 680
Cell pellet (from 5 mL of the original cell suspension in Percoll) washed with 71
80 mL of Eagle's MEM and centrifuged at 200 × g for 10 min
Washing repeated once 0
Cells from 2 mL of the cell suspension were seeded on a 6 cm Petri dish and 0
80% of the cells attached to the dish. After four washings with 5 mL portions of
Eagle's MEM, the cells were detached with 0.01% trypsin plus 0.25% EDTA.
(Original work by Pertoft et al. reproduced by kind permission)

26
Other methods
Chromatography by gel filtration on Sephacryl™ S-1000 Superfine will separate Percoll from larger particles 10
(e.g., subcellular particles), which are eluted in the void volume. Removal of Percoll from microsomal vesicles by gel A 280
filtration on Sephacryl S-1000 Superfine has been reported (275). The authors followed the elution pattern by assaying [ 125 ]-labeled Percoll
8
for the microsomal marker enzyme NADPH-cytochrome c reductase (Fig 18). The resulting microsomal fraction was
A 280
l(cpm × 10 -3)
examined by electron microscopy and found to be almost free from Percoll (less than 0.5% compared with the initial 5
VO Vt
6
sample).
Preliminary experiments using electrophoresis to separate lysosomes and viruses from Percoll have been reported (21),
4
but the methodology is difficult and results are often unpredictable (Pertoft, personal communication). 3
125
2
1
0
0 25 50 75
mL
1h
Fig 18. Gel filtration of microsomes obtained from gradients of Percoll containing 125I-labeled Percoll on
Sephacryl S-1000 Superfine. Vo = void volume, Vt = total volume (275, reproduced by kind permission of the
authors and publisher).
27
Practical notes
Care and cleaning of equipment
Polycarbonate tubes should be used with Percoll as the particles do not adhere to the walls of these tubes. Solutions of
Percoll usually produce a small pellet of compacted silica at the bottom of the tube after centrifugation and deposits on the
wall of tubing used for fractionation etc. These deposits may be difficult to remove when dry. Therefore, it is recommended
that all equipment is washed immediately after use. Spillages of Percoll can be removed by washing with water.
Storage of Percoll
Percoll can be stored unopened at room temperature for up to 5 years. When opened, it should be stored below 8°C.
If opened under non-sterile conditions, Percoll may be frozen for up to 6 months at -18°C (allowing sufficient headspace
for expansion) to avoid microbial growth. If stored at -18°C, gradients form upon thawing, necessitating a mixing of the
contents of the bottle before use. Preformed gradients can be stored for weeks without a change in gradient shape,
provided that the gradient is sterile and is not physically disturbed.
Sterilization of Percoll solutions

Autoclaving of Percoll solutions must be carried out in the absence of salts or sucrose (i.e., do not autoclave SIP).
When autoclaving undiluted Percoll, it is recommended that minimum contact with air be maintained to avoid particle
aggregation at the Percoll/air interface. This can be accomplished by using a narrow-necked bottle when autoclaving.
If these particles form, they may be removed by low speed centrifugation. If any significant evaporation occurs during
autoclaving, the volume should be replenished with sterile water so that the density is not affected.
Aggregates of silica particles

It is an inherent tendency of all silica colloids to form aggregates, either during autoclaving as described above, or
upon prolonged storage. These aggregates may be observed in some batches of Percoll either as a slight precipitated
sediment or as a faint white band which has a density of 1.04 to 1.05 g/mL. This band may form during gradient formation
in the centrifuge or during low speed centrifugation of a preformed gradient. The aggregated silica does not interfere
with the separation of biological particles as almost all cells and organelles have buoyant densities in Percoll of greater
than 1.05 g/mL.
28
Percoll PLUS — low endotoxin
Physical properties
Composition Colloidal silica solution with pH 9.4 ± 0.5 at 20°C
covalently linked silane
Osmolality < 30 mOsm/kg H2O Carbon content in dry residue 4.0 to 5.5%
Density 1.130 ± 0.005 g/mL Endotoxin activity (max) 2 EU/mL
Viscosity < 15 cP at 20°C Shelf life 5y
Composition
Percoll PLUS is a silica-based colloidal medium for cell preparation by density gradient centrifugation. It provides all
the advantages of Percoll and can be incorporated into existing procedures using Percoll gradients for the preparation
of a variety of human cell types. The silica particles of the medium are covalently coated with silane providing greater
product stability and low osmolality, toxicity, and viscosity.
Osmolality
Percoll PLUS has a low osmolality of < 30 mOsm/kg H2O and can easily be adjusted with physiological saline, other
balanced salt solutions, or cell culture media to give gradients that are iso-osmotic and adjusted to physiological
conditions throughout.
Density
Percoll PLUS is provided having a density of 1.130 ± 0.005 g/mL. After adjustment, Percoll PLUS forms iso-osmotic
gradients within the density range of 1.0 to 1.3 g/mL. This density range is optimized for separation of most cells,
subcellular particles, and larger viruses, which have a buoyant density of 1.0 to 1.2 g/mL in Percoll PLUS.
29
Endotoxin activity
The Percoll PLUS medium has low endotoxin levels (< 2 EU/mL). Low toxicity improves safety making Percoll PLUS
well-suited for cell separation in clinical research applications.
Gradients
Under moderate centrifugal force, the colloidal particles in Percoll PLUS medium sediment to form smooth, continuous
density gradients and this property can be exploited in either ﬁxed-angle or vertical rotors.
Percoll PLUS is also ideally suited to applications where high-speed centrifugation is required. In this case, the sample
can be pre-mixed with the medium and subsequently separated on the continuous gradient formed in situ. Thus, gradient
formation and sample separation can be achieved in one step.
Further details on centrifugation conditions and buoyant densities of cells, subcellular particles, and viruses centrifuged
on Percoll gradients can be found elsewhere in this handbook.
Practical notes
Storage
See page 28, Practical notes for Percoll.
30
Applications 5 min at 400 × g 15 min at 800 × g
Density g/mL
Number of cells
1010
9
10
Blood cells 1.10
ient
g r a d
The entire spectrum of cell types present in blood can be resolved on preformed gradients of Percoll. The method ty 10 8
Platelets Densi
described by Pertoft et al. (55) (Fig 19) utilizes both rate zonal (separation by size) and isopycnic (separation by density)
techniques. Diluted blood was layered on top of a preformed self-generated gradient and centrifuged for 5 min at 10 7
400 × g, during which time the thrombocytes or platelets (which are appreciably smaller than the other cells present)
did not penetrate into the gradient. 1.05 10 6
The plasma layer containing the thrombocytes was removed and replaced by saline, and centrifugation was
continued at 800 × g for 15 min, resulting in isopycnic banding of mononuclear cells (lymphocytes and monocytes), 0.5•106
polymorphonuclear cells and erythrocytes. The position and densities of the banded cells were monitored using MNC PMNC RBC
Density Marker Beads in an identical gradient contained in a second centrifuge tube.
1.00 0
Although the above method demonstrates the utility of Percoll for fractionating whole blood, most blood cells can be 1 2 3 4 5 6 7 8
appreciably enriched using a simple step gradient. A simple step gradient often gives acceptable yields and purity for Distance from the meniscus cm
downstream processing. The following Application tables contain a number of examples of purification of blood cells and 1 2 3 4 5 6 7 8 9 10
other cell types using different types of Percoll gradients. Volume mL
The following tables were complied to assist the researcher in selecting references most likely to contain relevant Fig 19. Separation of human blood cells in a gradient of Percoll. The tubes were filled with 10 mL of 70%
information regarding use of Percoll for a particular cell or tissue type. (v/v) Percoll in 0.15 M NaCl (p=1.086 g/mL), and the gradient performed by spinning in a 14° angle rotor
at 20 000 × g for 15 min. Two mL of gradient material was removed from the bottom of the tube using a
syringe, and 1 mL of heparinized blood diluted with 1 mL of 0.15 M NaCl was layered on top of the gradient.
Centrifugation was carried out as indicated. Densities were monitored using Density Marker Beads.
MNC = Mononuclear cells, PMNC = Polymorphonuclear cells, RBC = Red blood cells (55, reproduced
by kind permission of the authors and publisher).
31
Applications — blood cells
Lymphocytes
Species Gradient type Tissue type Comments Downstream application Ref. #
human continuous blood Percoll density centrifugation resulted in significant down-regulation of Immunoflourescence 891
L-selectin surface reactivity.
human continuous tonsil A Percoll density gradient was used for separation of large (low density) in vivo cell culture, FACS, granulocyte-macrophage colony stimulating factor 892
activated cells from small (high density) resting cells. (GM-CSF) assays, and Northern blots
human continuous spleen, tonsil Large B lymphocytes from tonsils (in vivo activated cells) obtained by Percoll cell culture, Northern blots, FACS 893
gradient centrifugation displayed higher IL-4R levels than resting cells.
human continuous peripheral blood Percoll was used to separate proliferating form nonproliferating cells. tritiated thymidine incorporation 11
human continuous tonsil, peripheral blood This procedure yielded > 90% viable cells and has proved quite helpful in proliferation and cytotoxicity assays 16
renewing overgrown cultures.
human continuous blood Percoll was used to separate monocytes from lymphocytes. cell culture, coagulation activity, immunoradiometric assays 40
human discontinuous (3-layer) intestine Lymphocytes were enriched in the interface between 66.7 and 44% Percoll. flow cytometric analysis, immunoperoxidase procedure, cell culture 894
Further purification was performed using magnetic beads.
human discontinuous (6-layer) peripheral blood Percoll was used to separate large granular lymphocytes (LGL) from peripheral detection of CD5LOW+ in the LGL population 895
mononuclear cells.
human discontinuous tonsil Percoll gradient was used for the separation of small (high density) and large cell culture, apoptosis assays, immunoassay for G-CSF, bioassay for 896
(low density) cells. GM-CSF, Northern blot analysis
human discontinuous intestine proliferation assays, measurement of cytotoxicity, H1 receptor binding 897
studies
human discontinuous peripheral blood Percoll was used for the isolation of low density cells. FACS, immunoflourescence, nonspecific esterase staining 898
human discontinuous peripheral blood Lymphoctes were recovered from low density Percoll fractions. suppression of NK-cell proliferation by freshly isolated monocytes 899
human discontinuous tonsil Percoll was used to isolate follicular dendrite cells (FDCs). cell sorting, B cell proliferation by FDCs 900
human discontinuous bone marrow Percoll was used to isolate leukemic cells from bone marrow. establishment of a leukemic cell line 901
32
Lymphocytes (continued)
human discontinuous peripheral blood After separation on Percoll, a virtually pure population of activated cells immunoflourescence and assay of phospholipid metabolism 902
was obtained, as estimated by the presence of the 4F2 marker and of the
transferrin receptor.
human discontinuous (1-step) blood Lymphocyte purity was > 99% and the population of monocytes was enriched induction and assay of lymphokine (IL-2)-activated killer (LAK) 903
82 to 90%. cell activity
human discontinuous (4-layer) blood Percoll was used for separation of large granular lymphocytic (LGL) cells Giemsa staining, cell activation with interleukin-2 (IL-2) 904
from T cells.
human discontinuous (4-layer) tonsil Percoll was used for B cell enrichment. flow cytometry 905
human discontinuous (5-layer) blood Large granular lymphocytes (LGL) were collected from the low density fractions, FACS, cell culture, cytotoxicity assays 906
whereas T cells were located in the higher density bottom fraction.
human discontinuous (5-layer) blood Monocytes were purified up to 90% and lymphocytes to > 99%. cell counting (hemocytometer) and cell culture assays 69
human discontinuous (7-layer) peripheral blood cytotoxicity assay, flow cytometry analysis, and 907
complement-dependent lysis
human self-generating peripheral blood Percoll was used to separate viable and nonviable cells. Yields were slightly cytotoxicity assays 83
higher and erythrocyte contamination was slightly lower with Percoll than
with Ficoll-Isopaque.
canine continuous blood Percoll was used for enrichment and depletion of antibody-positive cells. reverse hemolytic plaque assay and cell-mediated lympholysis 908
canine discontinuous whole blood A final sedimentation of purified lymphocytes through a 45/50% Percoll measurement of NK activity 909
(4-step and 2-layer) gradient concentrated natural killer (NK) activity into a single band of
lymphocytes.
mouse continuous (3-layer) intestine Enrichment increased from 44.1% (single filtration) to 52.4% (multiple filtration) flow cytometry 910
after nylon wool filtration, and from 70.3% (single filtration) to 82.8% (multiple
filtration) after Percoll fraction.
mouse continuous (5-layer) spleen Percoll was used for separation of virgin and memory T cells. cell proliferation assays, FACS 911
mouse discontinuous (3-layer) spleen Percoll was used for separation of B cells. protein phosphorylation assay 912
mouse discontinuous (3-layer) intestine Percoll was used for isolation of intestinal intraepithelial lymphocytes (IEL). DNA analysis by flow cytometry, mRNA-cDNA dot blots, PCR 913
mouse discontinuous (4-layer) spleen Percoll was used for isolation of small, resting B cells. cell cycle analysis by flow cytometry 914
bovine discontinuous mammary Purified cells were > 80% pure. Wright´s Giemsa staining, cell culture 915
33
Monocytes
human discontinuous peripheral blood With the Percoll minigradient, cells could be obtained in 90 to 100% from cytogenic analysis 916
(minigradient) the patients at all time points after bone marrow transplant (BMT).
human continuous blood The isolated mononuclear leukocyte (MNL) fraction contained > 80% cells cell culture 917
giving a positive reaction for a-naphthyl acetate esterase (a-NAE).
human continuous peripheral blood Percoll was used to isolate monocytes with > 85% purity and > 95% viability. cell culture with cytokines 918
human continuous blood Percoll has proved very practical for the separation of monocytes from blood cell culture 34
and of macrophages from ascites and synovial fluids.
human continuous blood Percoll gradients were used for the separation of monocytes from lymphocytes. cell culture 40
human continuous blood A one-step procedure was used for obtaining a high-yield suspension of cell counts, Fc-receptor presence and phagocytosis assays 57
monocytes of 20% purity, which does not require washing before cultivation.
A two-step method gave better than 90% pure monocytes at a lower yield.
human continuous peripheral blood MNL were separated into two fractions with Percoll: one consisting mostly fungal (Coccidioides immits) killing assay 919
of monocytes and the other lymphocytes.
human discontinuous blood Monocyte purity was 95%. cell culture 920
human discontinuous whole blood Percoll gradient was used for enrichment of hematopoietic progenitor cells. assay for colony formation 921
human discontinuous blood RNA isolation, Northern blot analysis and RT-PCR 922
human discontinuous bone marrow DNA hybridization studies 923
human discontinuous (1-layer) blood Lymphocyte purity was > 99% and the population of monocytes was induction and assay of lymphokine (IL-2)-activated killer (LAK) activity 903
enriched 82 to 90%.
human discontinuous (1-layer) blood PMN recovery was > 90% and RBC contamination < 5%. Northern blot analysis 924
human discontinuous (1-layer) peripheral blood Monocytes were ≥ 95% pure. Northern blot analysis, nuclear runoff experiments, 925
S1 protection assay
human discontinuous (4-layer) peripheral blood Cells obtained from the 65% to 75% interface were 99% granulocytes. analysis and Western blot analysis genomic DNA isolation and PCR 926
human discontinuous (1-layer) peripheral blood With the 1-step gradient, the purity of the monocytes was 93 to 96%. Giemsa staining and cell culture 927
34
Monocytes (continued)
human discontinuous (5-layer) peripheral blood Percoll-isolated monocyte/macrophages as identified by Wright-Giemsa stain. interactions between monocyte/macrophage and vascular smooth 928
muscle cells
human discontinuous (5-layer) blood Monocytes were purified up to 90% and lymphocytes to 99% purity. cell recovery counting and cell culture assays 69
human discontinuous peripheral blood cell enumeration with Coulter counter, RNA isolation, and Northern 929
blot analysis
equine discontinuous (1-layer) peripheral blood All MNCs were recovered on Percoll gradients without any neutrophil cell recovery assays 930
contamination.
Erythrocytes
human continuous whole blood Percoll was used for separating young and old erythrocytes. immunoflourescence analysis of complement receptor type 1 (CR1) 931
and CD59, proteolytic cleavage of CR1 in vivo
human continuous blood Percoll was used to separate Plasmodium falciparum-parasitized erythrocytes isolation of erythrocyte membranes lipid peroxidation, vitamin E and 932
from nonparasitized erythrocytes. transmembrane reducing system analysis
human continuous blood A rapid method for the age fractionation of human erythrocytes by Percoll flame photometry, enzyme assays 77
density gradient centrifugation was described.
human discontinuous (4-layer and blood A rapid method using Percoll to fractionate erythrocytes according to age analysis of the decline of enzymatic activity in aging erythrocytes 933
8-layer) was described.
human discontinuous (4-layer) blood ELISAs, proteolytic digestion of membranes 934
human discontinuous (4-layer) blood The position of Density Marker Beads was used to collect cells with densities yield stress experiment: a sensitive index of cell: cell adhesion 935
< 1.00 g/cm3 or > 1.119 g/cm3. of deoxygenated suspensions of sickle cells
human discontinuous blood Percoll gradient was used to separate erythrocytes into 4 density fractions. platelet-activating factor (PAF) acetylhydrolase activity and 936
membrane fluidity
35
Erythrocytes (continued)
human discontinuous blood Erythrocytes loaded with L‑asparaginase using a hypotonic dialysis process L-asparaginase activity 937
were separated into eight fractions.
human discontinuous blood Discontinuous gradient of the range 1.080 to 1.115 g/cm3 with each layer enzyme assays 66
differing in density by 0.005 g/mL produced nine cell fractions.
human discontinuous (5-layer) blood study of RBC deformability and cell age 938
human discontinuous (8-layer) blood Percoll was used for density separation of RBC loaded with inositol haemoglobin distribution, distribution of IHP concentrations 939
hexaphosphate (IHP) by reverse osmotic lysis.
human discontinuous (9-layer) blood A detailed comparison between two cell-loading techniques for inositol oxygen affinity, hematological parameters and organic phosphate 940
hexaphosphate was performed by monitoring the RBC distribution patterns content measurements
on Percoll density gradients.
Mastomys continuous blood Percoll was used to separate Plasmodium berghei-parasitized erythrocytes cAMP level in RBCs 941
natalensis from non parasitized cells.
mouse continuous (self-forming) blood Fractionation of RBC yielded five distinct populations that maintained their transbilateral movement and equilibrium distribution of lipid 942
densities upon recentrifugation in a second gradient.
mouse continuous peripheral blood Percoll was used for density gradient separation of chemically-induced fixing, staining and flow cytometric analysis of micronucleated 943
erythrocytes. polychromatic (MPCE) and micronucleated nonchromatic (MNCE)
erythrocytes
mouse discontinuous peripheral blood Erythrocytes were contaminated with only 0.001% nucleated cells. glucose phosphate isomerase (GPI) assay 944
rat discontinuous whole blood Percoll was used to separate Plasmodium berghei-infected RBCs. oxygen dissociation analysis 945
rabbit discontinuous (7-layer) blood Rabbit red blood cells were reproducibly fractionated into populations of measurement of cytosolic protease activities 946
various stages of maturation.
trout discontinuous blood The gradient in the region of 45 to 65% Percoll produced three red cell antioxidant enzyme activities and membrane fluidity analysis 947
fractions which is due to multiplicity of haemoglobin components.
36
Natural Killer (NK) cells
human discontinuous peripheral blood The Percoll (preculture) step facilitated the density separation of resting cells NK- and T-cell activation, immunoflourescence 948
from larger lymphocytes.
human discontinuous blood K562 cells which adhere to NK cells were separated together. Enrichment of cytotoxicity studies, morphological characterization 61
NK cells was 71.3%.
human discontinuous (2-layer) peripheral blood The low density fraction (42.5 to 47.5% Percoll) which showed a 4-fold NK activity and kinetic constant determinations, measurement 949
enrichment in NK activity was used. of the effect of divalent cations on NK activity, and effect of ATP
on NK cell-surface markers
human discontinuous (6-layer) blood Further purification using magnetic beads resulted in a pure preparation. cytotoxic assay 950
human discontinuous (8-layer) peripheral blood Recovery was > 80% while viability, as judged by trypan blue exclusion, NK cell stimulatory effect, phenotype evalution by 951
was > 95%. immunoflourescence
mouse discontinuous (3-layer) lung The cells at the 50/55% interface were the richest in NK cell activity. adoptive transfer to reconstitute NK activity in NK-depleted mice 952
mouse discontinuous (6-layer) spleen NK cells were enriched in the lower density Percoll fraction, while natural cytotoxicity of NK cells was measured 953
cytotoxic T cells (NCT) were distributed between both higher and lower
density fractions.
mouse discontinuous (6-layer) liver All NK activity was above 1.08 g/mL density. Interfaces at 1.04 and 1.06 gave PCR, Western blot analysis, and cytotoxicity assays 954
a 2× enrichment of NK progenitors.
37
Neutrophils
human discontinuous (1-layer) whole blood Neutrophils pelleted in the 1.077 g/mL cushion. FACS analysis, intracellular Ca++ and superoxide anion measurements 955
human discontinuous (2-layer) whole blood polymorphonuclear neutrophil (PMN) labeling by 956
immunoflourescence, adherance assay and superoxide assay
human discontinuous (4-layer) peripheral blood Percoll was used to separate monocytes and lymphocytes. immunoflourescence and flow cytometry 957
human discontinuous (4-layer) whole blood Eosinophils and neutrophils were isolated following dextran sedimentation. flow cytometry and measurement of lactoferrin release 958
human discontinuous blood Cell preparation was layered onto a Percoll cushion to remove monocytes. immunoflourescence studies 959
After lysis of the erythrocytes, primarly neutrophils, with the remaining cells
being predominantly eosinophils.
human discontinuous blood The neutrophils were > 95% pure. indirect immunoflourescence, immunoelectron microscopy and FACS 960
analysis, O2 consumption
human continuous, nonlinear blood Percoll was used for subcellular fractionation of azurophil granules, specific ELISAs for NGAL, gelatinase, lactoferrin and myeloperoxidase 961
(2-layer) granules, gelatinase granules, plasma membranes, and secretory vesicles.
mouse continuous peritoneum An ~97% pure polymorphonuclear neutrophilic leukocyte (PMN) preparation electrophoretic analysis, GM-CSF assay, and cell morphology 70
was obtained using Percoll. and counts
38
Eosinophils
human discontinuous peripheral blood Eosinophils were purified using Percoll gradients followed by immuno-magnetic FACS analysis, eosinophil migration assays, Ca++ measurements 962
beads. Using this procedure, the eosinophil purity was always > 95% and the
viability was > 98%.
human discontinuous (2-layer) blood The recovery of eosinophils was 40% to 60%, the viability > 98% as tested by chemotaxis and intracellular Ca++ measurements 963
trypan blue exclusion, and the purity > 85%.
human discontinuous (2-layer) blood Eosinophil purity was > 95%, and the method did not induce priming of the serum-treated Zymosan (STZ) binding and placenta-activating factor 964
eosinophils. (PAF) measurments
human discontinuous (2-layer) blood Eosinophil purity was always > 85% and the recovery ranged from 40% to 60%. chemotaxis assay 965
Viability was > 98%.
human discontinuous (3-layer) peripheral blood Eosinophil purity was 95% to 99%, viability using trypan blue was > 98%, and density distribution analysis, cell culture 966
recovery was 40% to 60%.
human discontinuous (4-layer) whole blood The effect of dextran sedimentation on the density of neutrophils and flow cytometry and measurement of lactroferrin release 958
eosinophils was analyzed.
39
Basophils
human continuous peripheral blood Basophils were purified by Percoll density gradient separation and cell flow cytometry, histamine release, electron microscopy 967
sorting. The procedure yielded 95% purity with a total yield estimated to
range from 5% to 28%.
human continuous bone marrow The purity of basophils in the low density fraction (< 1.063 g/mL) was histamine content and release 968
generally > 75% of the cells.
human discontinuous peripheral blood Highly purified basophils were obtained by Percoll gradient followed by effects of cytokines on human basophil chemotaxis 969
negative selection using flow cytometry.
human discontinuous (2-layer) blood The majority of the basophils were located at the 1.070 to 1.080 interface. further purification by negative selection using immuno-magnetic 970
The purity in this fraction was 36% to 63%. beads
human discontinuous (2-layer) blood Highly purified basophils were obtained by Percoll gradient followed by histamine release assay, chemotactic assay 971
negative selection using flow cytometry.
human discontinuous (3-layer) whole blood Basophils were purified to > 80% using Percoll gradient followed by treatment flow cytometry and leukotriene C4 generation following calcium 972
with monoclonal antibodies to remove contaminants. ionophore stimulation
human discontinuous (3-layer) peripheral blood Basophil purity was 85% to 96% using Percoll. cell stimuli and mediator release assay 973
rat discontinuous blood further purification by immuno-magnetic beads, 974
immunoflourescence, electron microscopy
40
Applications — other cell types
Liver cells
human continuous liver Purification of cryo-preserved hepatocytes on Percoll density gradients primary cell culture, electron microscopy, viability assay radiolabeled 975
increased the percentage of viable cells from 55% to 87%. protein synthesis, secretion assay, metabolic studies, toxicological
studies
rat continuous liver Percoll offered a good way to obtain an enriched population of Kupffer cells. peroxidatic reaction 20
Recovery was 82%, viability 87% and purity 67%.
rat continuous liver Percoll gradients were used to isolate hepatocyte plasma membranes and phase contrast microscopy, cell binding experiments 33
mitochondrial membranes.
rat continuous liver Rat liver cells furnished subpopulations of parenchymal cells (hepatocytes) cell culture 55
having buoyant densities of 1.07 to 1.09 g/mL, and non-parenchymal cells
(mostly phagocytosing Kupffer cells) at a density of 1.04 to 1.06 g/mL.
rat NA liver Final preparations contained less than 5% nonviable cells as judged by trypan cell culture 71
blue exclusion.
rat continuous liver Percoll gradients were used to franctionate nonparenchymal cells into Kupffer light and flourescence microscopy, carboxyesterase and 976
cells, stellate and endothelial cells. Glutathione-S-transferase (GST) activities
rat discontinuous (2-layer) liver Percoll provided a simple, low cost, and rapid method for the isolation, electron microscopy, cell culture, trypan blue exclusion 977
purification and cultivation of rat liver sinusoidal endothelial cells (LEC).
rat discontinuous (2-step) liver Percoll gradients were used to separate fat storing cells (FSC) from liver cell culture 978
endothelial cells (LEC) and Kupffer cells (KC).
rat continuous liver Following the removal of damaged cells by centrifugation in Percoll, the mean cell viability and study of xenobiotic metabolism 979
viability of cryo-preserved hepatocytes, tested by trypan blue exclusion, was
88.6% (±1.3%).
rat continuous liver Percoll was used to remove dead cells from cryopreserved cells. Cell viability cell viability and study of xenobiotic metabolism 980
was 88 ±1% after the Percoll step.
41
Liver cells (continued)
rat continuous liver If cryo-preserved cells were purified by a Percoll centrifugation after thawing, Lowry protein assay, cytochrome assay, enzyme assays 981
the enzyme activities were not significantly different from those of freshly
isolated parenchymal cells, and the viability was 86%.
rat continuous liver Percoll separation yielded cryo-preserved cells with a viability and metabolic protein determination, enzyme assays and metabolism of 982
capacity not measurably different from freshly isolated cells. testosterone and benzo(a) pyrene (BaP)
rat discontinuous (2-layer) liver Percoll two-step gradients were used to separate Kupffer cells (KC) and liver light microscopy, electron microscopy and peroxidase staining 983
endothelial cells (LEC). Preparations of KC were 85% to 92% homogenous while
the LEC preparation was at least 95% pure.
rat discontinuous (5-layer) liver, spleen Percoll gradients were used to separate both spleen and liver cells. Spleen trypan blue viability assay, cell culture 984
and liver cell viability was over 95%.
rat continuous liver biopsy Percoll was used for separation of hepatocytes and non-parenchymal cells, cell enumeration using Coulter counter, immunocytochemistry, 985
as well as subfractionation. DNA extraction, Southern blot analysis, assay of marker enzymes
and protein in subcellular fractions, electron microscopy
42
Leydig cells
human continuous testis Percoll-purified Leydig cells were 70% to 80% pure based on staining for cell culture, stimulation of testosterone production 986
3 beta-hydroxysteroid dehydrogenase.
human continuous testis Percoll-purified Leydig cells were 80% to 90% pure as determined by immunocytochemical localization of apolipoprotein E (apoE) 987
3 beta-hydroxysteroid dehydrogenase staining.
human discontinuous (4-layer) testis Percoll gradients were used to isolate human Leydig cell mesenchymal precursors. cell culture 988
human discontinuous (5-layer) testis Percoll gradient centrifugation permitted isolation of two Leydig cell fractions. cell culture 989
mouse continuous (linear) testis Two groups were obtained: group 1 had densities of 1.0667 to 1.0515 g/mL; in vitro testosterone production electron microscope stereology 990
group 2 had densities of 1.0514 to 1.0366 g/mL.
porcine discontinuous testis Purity of Leydig cells was > 85%. effect of hydrocortisone (HS) and adrenocorticotropic hormone 991
(ACTH) on testosterone production
rat continuous testis Rat Leydig cells were purified from testis using elutriation followed by Percoll cell culture, the effect of human chorionic gonadotropin (hCG) on its 992
gradient centrifugation. gene regulation and protein secretion
rat continuous testis cell culture, the effect of GH-releasing hormone (GHRH) on Leydig cell 993
steroidogensis
rat continuous testis Rat Leydig cells were purified from testis using elutriation followed by Percoll cell culture in presence of 125I-labeled hCG, testosterone and cAMP 994
gradient centrifugation. Band 2 (of 3) contained > 95% Leydig cells (average production
density was 1.075 g/mL).
rat continuous testis Comparison of Leydig cells of different densities were made. viability staining, cell culture 995
rat continuous testis viability staining, in vitro testosterone production, SDS-PAGE 996
electrophoresis
rat continuous testis Isolation by Percoll gradient resulted in complete retention of morphological cell culture in presence of human chorionic gonadotropin (hCG), 31
and biological integrity and a purity of 90 to 95%. phase contrast microscopy, light microscopy and electron microscopy
rat discontinuous (2-step) testis cell culture in the presence of interleukin-1 (IL-1) 997
rat continuous testis Leydig cell precursors and pure (96%) Leydig cells were isolated on Percoll cell culture in presence of human chorionic gonadotropin (hCG) 998
(self-generating) gradients.
rat discontinuous testis The purity of Leydig cells ranged from 90% to 95%. cell culture in presence of human chorionic gonadotropin (hCG) 999
rat discontinuous and testis In the discontinuous gradient, the densest fraction contained a high proportion I-labeled iododeoxyuridine incorporation
125
1000
continuous of Leydig cells whereas the lighter fraction contained mostly non-Leydig cells.
43
Spermatozoa
Species Gradient type Comments Downstream application Ref. #
bovine discontinuous Percoll was thought to improve semen and preserve acrosome integrity. acrosome microscopy evaluation 1024
hamster continuous Caput epididymal spermatoazoa, with a specific gravity of 1.10–1.12 g/mL, lipid extraction and fractionation electron microscopy 1025
were isolated without contamination by other cells.
macaque continuous Percoll separation resulted in increased sperm-zona binding and did not affect zona binding experiments, acrosome reaction, motility assays 1026
the percentage of acrosome-reacted sperm bound to the zona or the percent
motility and percentage of acrosome-reacted sperm in suspension.
44
Bone marrow cells
normal human discontinuous (2-layer) bone marrow Megakaryocytes were at the interface between 1.020 g/mL and 1.050 g/mL. magnetic beads for further purification, flow cytometry 1027
normal human discontinuous blood B cells were recovered at least 95% pure. Gradients removed B-cell blasts flow cytometry 1028
very effectively.
HIV infected, discontinuous (2-layer) bone marrow Cells at the 1.020/1.050 interface were enriched 10-fold in megakaryocytes, megakaryocyte cultures prepared from immature cells for in situ 1029
normal and while those at the 1.050/1.070 interface were immature cells. hybridization
immune throm-
bocyto-penic
purpura human
normal human discontinuous (2-layer) bone marrow Percoll density fractionation resulted in the depletion of greater than 95% preparation of RNA and subsequent PCR, flow cytometry 1030
of total marrow cells and an increase in megakaryocyte frequency from
about 0.05% to 3% to 7%.
normal and discontinuous (3-layer) bone marrow Cells prepared were suitable for cell culture. colony plaque assay, immunoflourscence, flow cytometry, protein 1031
arthritic human colony blotting, RNA-colony blotting
normal and discontinuous (4-layer) peripheral blood Low density cells post- and pre-transplant were prepared for analysis. magnetic beads for further purification, PCR 1032
leukemic human
normal human discontinuous (7-layer) bone marrow T cells obtained using Percoll were enriched about two-fold in the high-density flow cytometry, mixed lymphocyte reaction assay, natural killer cell 1033
fractions of marrow cells and depleted by about four- to five-fold in the assay, cell culture
lowest-density fraction as compared with Ficoll™ medium.
normal human discontinuous (1-layer) bone marrow Bone marrow cells were prepared using Percoll to remove RBC. isolation of CD34+ cells using soybean agglutinin-coated flasks, 1034
progenitor cell assays, and flow cytometry
marmoset discontinuous (1-layer) bone marrow Bone marrow megakaryocytes from both interleukin-6 (IL-6) treated and flow cytometry 1035
untreated animals could be separated in Percoll.
primate discontinuous (1-layer) bone marrow Bone marrow was isolated from both normal monkeys and interleukin-6 (IL-6) cell enumeration, FACS, digital imaging microscopy and electron 1036
treated monkeys. microscopy
45
Bone marrow cells (continued)
monkey discontinuous (1-layer) bone marrow peripheral Light density cells were prepared from aspirates over a 60% cushion. cell culture and identification of various colony types 1037
and blood
mouse discontinuous (1-layer) bone marrow Red blood cells were removed from bone-marrow preparations with a single culture of hematopoietic precursers, effects of interleukin-10 (IL-10) 1038
70% Percoll cushion. on proliferation, alkaline phosphatase activity, collagen synthesis
assay, osteocalcin, preparation of RNA, and electron microscopy
mouse discontinuous (3-layer) bone marrow Bone marrow progenitor cells were suitable for culture. effects of interleukin-3 (IL-3) and lipoplysaccharide (LPS) on 1039
cultured cells
mouse discontinuous (3-layerlayer) bone marrow Cells prepared were depleted of lymphoid and macrophage-lineage cells by FACS analysis, hematopoietic progenitor cell culture, reconstitution of 1040
addition of monoclonal antibody plus complement. lethally irradiated mice
mouse discontinuous (3-layer) bone marrow Percoll was used to separate bone marrow fractions containing mostly blasts FACS analysis, chemotaxis assay, assay of colony-forming 1041
and lymphoid cells from those containing a high level of colony-forming units-spleen (CFU-S)
units-spleen (CFU-S) counts.
mouse discontinuous (3-layer) protease-treated calvarial Percoll gradients gave distinct subpopulations of cells based upon the results primary cell culture, flow cytometry, insulin-like growth factor I (IGF-I) 1042
bone sections of various assays. assay, binding of epidermal growth factor, alkaline phosphatase
determination
mouse discontinuous (4-layer) bone marrow Normal suppressor cell activity was maintained after separation. suppressor cell activity assay 1043
mouse discontinuous (4-layer) bone marrow Cells at a 1.06/1.07 g/mL density were used in subsequent studies. reconstitution of lethally irradiated animals 1044
mouse discontinuous (5-layer) bone marrow, spleen flow cytometry, reconstitution of lethally irradiated mice 1045
rat discontinuous (3-layer) bone marrow About 75% of the input CFU-megakaryocytes (CFU-MK) were recovered in culture of hematopoietic progenitor cells 1046
the fraction between 1.063 and 1.082 g/mL Percoll. CFU-MK were enriched
only in this density fraction.
rabbit continuous bone marrow implantation into in vivo placed diffusion chamber, cytochemical 38
staining, and electron microscopy
feline discontinuous (1-layer) bone marrow Marrow mononuclear cells from both feline immunodeficiency virus-infected culture of hematopoietic progenitor cells 1047
cats and normal cats were isolated.
46
Macrophages
human discontinuous lung Alveolar macrophages were purified from contaminating granulocytes using superoxide (SO) release 1048
a discontinuous Percoll gradient.
human discontinuous (4-layer) brochoalveolar lavage Percoll gradients gave > 95% alveolar macrophage (AM) purity. cell viability assay, light microscopy 1049
human discontinuous (4-layer) lung Use of Percoll resulted in near total purification of alveolar macro-phages (AM) superoxide (SO) anion release 1050
from other cells.
human discontinuous (4-layer) decidual tissue When cells were purified further with Percoll, the percentage of CD-14-positive secretion of platelet-activating factor (PAF) acetylhydrolase 1051
cells increased by 52%.
human discontinuous pulmonary > 97% of the cells of fractions 1 to 4 were (4-layer) shown to be alveolar nonspecific esterase staining, flow cytometric DNA analysis 1052
macro-phages (AM) in a previous study.
human discontinuous (4-layer) lung This method was used to study alveolar macrophage (AM) heterogeneity. cell viability, esterase and peroxidase activity assays, electron 1053
The increased numbers of hypodense AM found in the asthmatic patients microscopy, generation of superoxide anion and thromboxane B2
were unlikely to be due to the procedure.
human discontinuous (5-layer) peripheral blood Percoll-isolated monocyte/macro-phages were harvested from the top layer interactions between monocyte/macrophage and vascular smooth 928
and routinely contained 75%/90% monocytes/macrophages as identified by muscle cells
Wright-Giemsa stain.
mouse continuous and peritoneum The total cell yield was 100.0% ±0.8%, and as measured by the trypan blue light microscopy, trypan blue exclusion, esterase activity assay, 1054
discontinuous exlusion test, the cell viability was completely preserved. peroxidase activity assay, cell immunophenotyping, bacterial
phagocytic assays
mouse discontinuous (4-layer) cultured cells Percoll did not have a detectable effect on the cytolytic activity of cultured phagocytic and cytolytic assays 30
macrophages or on their viability.
mouse, rat continuous and peritoneum A continuous gradient followed by a discontinuous gradient was used to isolate trypan blue exclusion 1055
discontinuous all cell populations according to their actual density. This procedure yielded
cells of high viability with preservation of critical cell function.
rat discontinuous (5-layer) lung The Percoll fractions were designated I to IV in order of increasing density fluorescence microscopy 1056
with a percent distribution of cells of about 5%, 15%, 50% and 30%, respectively.
Cell viability was > 95%.
47
Macrophages (continued)
rat discontinuous (5-layer) lung Cell viability was > 95% by trypan blue exclusion and > 95% were identified effects of pulmonary surfactant and protein A on phagocytosis, 1057
as alveolar macrophages (AM) in un-fractionated and fractionated cells by light microscopy
Giemsa and nonspecific esterase stains.
rat continuous broncho- alveolar lavage The various fractions comprised approximately 90% to 99% macrophages in esterase activity, surface expansion of Ia antigen by an 1058
virtually all instances. immunoperoxidase technique
Mast cells
mouse NA peritoneum Purity of the mast cells was nearly 100%, as checked by Memacolor fast qualitative and quantitative PCR analysis 1059
staining.
mouse continuous peritoneum Starting from a peritoneal cell population containing 4% mast cells, electron microscopy and ultrastructural cytochemistry 8
a mast cell purification of up to 95% was obtained.
rat discontinuous peritoneum Mast cell purity with Percoll was > 95%. direct interaction between mast and non-mast cells, histamine 1060
release assay
rat continuous peritoneum Mast cells purified on Percoll gradients were more than 90% pure by toluidine flourometric assay to measure histamine release 1061
blue staining, and the viability was > 98% by the trypan blue exclusion test.
rat continuous peritoneum Mast cells can be isolated with high yields and purity by centrifugation on light and electron microscopy, cytofluorometry 9
gradients of Percoll.
rat continuous (sequential) peritoneum The purity of mast cells purified over sequential Percoll gradients was histamine release and peroxidase activity 1062
evaluated by measurement of the contribution of eosinophil peroxidase
to mast cell peroxidase activity.
48
Thymocytes
mouse discontinuous (5-layer) thymus Percoll was used for separation of immature thymocytes. in vitro stimulation by mitogens, isolation of nuclei, isolation and 1063
gel electrophoresis DNA, enzyme assays
rat discontinuous thymus Percoll was used for separation of normal and apoptotic thymocytes. flow cytometry 1064
rat discontinuous (4-layer) thymus Percoll was used for separation of cells possessing the characteristically electron microscopy, Coulter counter analysis, flow cytometry, 1065
condensed nuclear chromatin associated with apoptosis from apparently DNA analysis
normal thymocytes.
rat discontinuous (4-layer) thymus Percoll was used for isolation of a transitional population of pre-apoptotic DNA analysis, isolation of nuclei and DNA autodigestion, light and 1066
thymocytes. electron microscopy
rat, mouse discontinuous (3-layer) thymus Percoll was used to separate large and small thymocytes. An extremely phase contrast microscopy and autoradiography 62
high level of viability was maintained.
Miscellaneous cells
Cell type Species Gradient type Tissue type Comments Downstream application Ref. #
pancreatic islets human, mouse continuous pancreas The use of Percoll eliminated the problems of high viscosity, undesired density determination and insulin secretion 5
osmotic properties and, in some cases, also toxic effects.
endothelial human continuous linear whole blood Final recovery of endothelial cells was 91.6%. immunofluorescence 1067
gradient
trophoblasts rat continuous placenta Percoll gradient centrifugation yielded efficient separation of rat placental development of in vitro rat placental trophoblast cell culture system 1068
lactogen-II (rPL-II) producing cells from digested tissue from labyrinth and
junctional zones of the chorioallantoic placenta.
various NA NA NA This paper compared different approaches to cell separation. According none 1069
to the authors, Percoll is generally the most useful media for isopycnic
centrifugation of most kinds of cells.
49
Miscellaneous cells (continued)
viable vs. nonviable human, rat discontinuous various tumor Interface showed a viability of > 90%, but the yield of viable cells decreased trypan blue viability assay, 2-D PAGE 1070
(2-layer) tissue dramatically if the tissue resection was not immediately processed.
apoptotic human discontinuous promyelocytic The step gradient used generated three main cell bands and a cell pellet, DNA isolation 1071
(7-layer) leukemic cell line the pellet was very enriched for apoptotic cells (85% to 90%).
lymphoblast human continuous whole blood Lymphoblasts were enucleated using a Percoll gradient containing electrofusion 1072
cytochalasin B.
brain capillary rat continuous brain Subsequent Percoll gradient centrifugation resulted in a homogenous cell culture, light microscopy electron microscopy 170
endothelial pre-made population of capillary endothelial cells capable of attachment to collagen
and incorporation of tritiated thymidine.
neurons rabbit discontinuous and dorsal-root ganglia Neurons were isolated with a viability of 80% and a purity of > 90%. cell culture, light and electron microscopy 480
rate zonal
non-myogenic chicken discontinuous breast muscles Separation of cells from embryonic muscle allowed direct analysis of cell culture, microscopy, DNA/protein analysis 680
separated from cell-specific proteins without the need for cell culturing.
myogenic
megakaryocytes human discontinuous bone marrow Isolation of megakaryocytes was reproducibly better in Percoll than in BSA. Ficoll 400 centrifugation to further purify, complement receptor assay 155
chondrocytes rat discontinuous bone marrow Cell viability was > 95% while yield varied depending on aggregation of cells. cell culture, quantitation of proteoglycans and collagen 629
spermiophages turkey discontinuous sperm Spermiophages fixed immediately after Percoll isolation resembled those light and electron microscopy, cell culture 1073
in freshly ejaculated semen except for an apparent increase in the number
of mitochondria.
NA human continuous parathyroid gland Densities of parathyroid glands were measured using various density gradient glandular density determination 2
media. For densities > 1.0 g/mL, Percoll proved superior to any of the other
gradient liquids investigated.
50
Applications — microorganisms
Microorganisms
Species Type Gradient type Host tissue Comments Downstream application Ref. #
Bacteroides sp. bacteria discontinuous NA Percoll was used to assess the degree of capsulation of the twelve Bacteroides light microscopy 1074
(4-layer) strains grown in three different media.
Ehrlichia ristcii bacteria continuous cultured cells Percoll was used to purify Ehrlichia risticii from an infected murine macrophage CO2 production assay, Coomassie brilliant blue dye binding assay 1075
cell line (P388D).
Ehrlichia risticii bacteria continuous cultured cells Ehlichia risticii was purified from an infected murine macro-phage cell line CO2 production assay, Coomassie brilliant blue dye binding assay 1076
(P388D).
Porphyromonas bacteria continuous NA Percoll was used to separate unbound cells from saliva-coated bead binding and binding inhibition assays 1077
gingivalis (SHAP)-bound cells.
Treponema bacteria continuous NA Percoll-purified treponemes from 5-day infections were immobilized influence of different sera on in vitro immobilization of Percoll-purified 1078
pallidum significantly more slowly than the purified trepo-nemes from 7- and Treponema pallidum
8-day infections.
Theileria sp. bacteria discontinuous bovine A purification method for viels from Theileria-infected bovine erythrocytes light and electron microscopy and 1- and 2-D poly-acrylamide gel 1079
(2-layer) erythrocytes was developed. electrophoresis
Babesia bigemina protozoa continuous and bovine Babesis bigemina-infected erythrocytes were successfully concentrated enzymatic studies and starch gel electrophoresis 1080
discontinuous erythrocytes at least 20 times by Percoll and Percoll-Renografin density gradients.
(4-layer)
Babesia equi protozoa continuous horse The piroplasms of Babesia equi were purified by lysis of infected horse protein characterization of B. equi piroplasms 1081
erythrocytes erythrocytes and Percoll density-gradient centrifugation.
Plasmodium protozoa continuous mouse blood Percoll was used for the separation of host erythrocyte membrane from electron microscopy, electro-phoresis, immuno-blotting, marker 1082
berghei and malarial parasites. The recovery of the erythrocyte membranes was enzyme analysis and pulse chase analysis
P. chabundi ~65% to 70%, whereas parasite re-covery was 80% to 90%, and the
relative purity was ~85% to 90%.
Babesia bovis protozoa continuous bovine A 65% Percoll concentration was found to be optimal for Babesia bovis parasite viability assay 1083
erythrocytes merozoite (i.e., mature exoerythrocytic stage) separation. A 100% Percoll
stock solution was optimal for enrichment of infected erythrocytes.
51
Microorganisms (continued)
Species Type Gradient type Host tissue Comments Downstream application Ref. #
Entamoeba protozoa discontinuous faecal cyst Percoll purification provided a good yield even from a moderate faecal cyst E. histolytica for use as antigen 1084
histolytica (2-layer) load in a single stool sample.
Vairimorpha protozoa continuous caterpillar Percoll was used to purify spores. 40% of the original spores were recovered infection of cultured cells 1085
necatrix with nearly all refractile (90% or more). Contaminating bacteria were not seen.
rice transitory virus continuous rice plant leaf Typical purification runs gave about 140 to 850 mg of purified virus per 100 g Lowry protein assay, electron microscopy, SDS-PAGE, ELISAs, 1086
yellowing virus of infected material. Western blots
(RTYV)
Rubivirus virus continuous cultured cells Comparison of Percoll and sucrose gradients for purifying Rubella gave a yield hemagglutinating titer assays 1087
(rubella virus) of 72% with Percoll compared to 8.6% with a sucrose gradient.
Herpes simplex virus continuous NA Percoll was used to purify herpes simplex virus. none 56
virus
dino-flagellates, marine continuous NA Most of the marine species recovered were in a condition that would permit light microscopy, motility assay, photosynthesis assay 60
diatoms, micro-algae direct physiological measurements of photosynthesis, respiration, ion
blue-green adsorption and specific growth rates.
bacteria
mycoplasma-like NA discontinuous lettuce (Lactuca Electron microscopy showed a high concentration of MLOs with well-preserved electron microscopy, ELISA 1088
organism (MLO) sativa) cellular structures.
52
Applications — subcellular particles
Plasma membranes
Species Gradient type Tissue/Cell type Comments Downstream application Ref. #
human continuous platelets A method for rapid isolation of platelet plasma membrane was described, radioactive tracer studies, enzyme and protein assays 54
(self-generating) based on the use of [3H]‑concanavalin A as a membrane marker and
self-generating gradients of Percoll.
rat, human continuous liver biopsy Plasma membrane enzymatic marker and membrane transport assays membrane enzyme assays and measurement of amino acid transport 1089
indicated that isolated membranes retained their functional integrity. by membrane vesicles
rat continuous uterus The plasma membrane markers, 5’-nucleotidase and cholesterol, were Ca++ uptake and release assays enzyme assays, cholesterol and 1090
enriched in the fractions near the top of the gradient, while the sarcoplasmic progesterone assays, and Western blot
reticulum marker enzyme, rotenone-insensitive NADH-cytochrome-c
reductase, was in the lower part.
rat continuous (3-layer) brain Synaptic plasma membranes were prepared by Ficoll and Percoll density phospolipase C assay, marker enzyme assays 1091
gradients.
rat discontinuous (2-layer) cultured cells Two subcellular fractions, one enriched in plasma membranes and the other electron microscopy, determination of enzymatic markers, enzyme 1092
enriched in endoplasmic reticulum membranes, were obtained by Percoll activity, calcium uptake and release
gradient fractionation.
rat continuous liver The plasma membrane marker, 5’-nucleotidase, was enriched, whereas the vesicle amino acid transport assay 1093
cytosolic (endoplasmic reticulum) enzyme, glucose-6-phosphatase, was
impoverished, indicating vesicle purity.
rat continuous liver Percoll gradients were used to isolate hepatocytes, plasma membranes and phase-contrast microscopy, cell binding experiments 33
mitochondrial membranes.
rat continuous liver Use of Percoll for the low speed nuclear pellet resulted in plasma membrane marker enzyme determinations, Ins(1,4,5)P3 binding, Bradford protein 1094
markers and Ins (1,4,5)P3 binding activity being purified together. assay, SDS-PAGE
rat continuous liver Percoll purified hepatic plasma membranes were used to examine the arginine transport activity, enzyme marker assays 1095
transport of amino acids.
53
Plasma membranes (continued)
Species Gradient type Tissue/Cell type Comments Downstream application Ref. #
bovine continuous cultured aortic Plasma membranes were labeled with trace amounts of [3H]‑cholesterol and enzyme assays and SDS-PAGE/ligand blots 1096
endothelial cells cell homogenates were fractionated on sucrose and Percoll gradients.
bovine discontinuous (3-layer) adrenal gland The procedure provided a fraction rich in plasma membranes. solubilization of plasma membranes, affinity chromatography, 78
radiolabeling of plasma membrane, enzyme assays
sheep continuous perirenal fat adipocytes The fatty acid content of plasma membranes was analyzed. fatty acid analysis using gas-chromatography 1097
(self-generating)
Chinese hamster continuous cultured chinese hamster A procedure yielded plasma membrane fractions that were enriched 3-fold lipid analysis, enzyme assays 1098
(self-generating) ovary (CHO) cells and practically free of lysosomes; pure endoplasmic reticulum (ER) and
mitochondrial fractions were obtained as well.
skate (Raja continuous liver Marker enzyme studies indicated that plasma membranes isolated with enzyme assays, fluorescence anisotropy measurements, alanine 1099
erinacea) (self-generating) Percoll gradients were highly enriched in the basolateral domain of the liver transport, protein and lipid determination
plasma membrane and largely free of contamination by intracellular organelles
or canalicular membranes.
Fungi (Penicillium continuous NA The majority of contaminating membranes were removed by Percoll enzyme assays, electron microscopy, membrane fusion, transport 1100
chrysogenum) step gradients. studies, Lowry protein assay
Fungi (Penicillium continuous NA Right-side-out plasma membrane vesicles were prepared using two-phase ATPase activities, electron microscopy 1101
cyclopium) partitioning and Percoll gradients.
54
Lysosomes
human continuous cultured fibroblasts Only lysosomes, sedimented in the bottom third of 30% to 40% Percoll adenosine deaminase and N-acetyl-b-hexosaminidase assays 1102
density gradients.
human continuous cultured fibroblasts A crude mitochondrial lysosomal pre-paration of fibroblasts was separated enzyme assays, SDS-PAGE electrophoresis, immunoblotting 1103
into high-density fractions (lysosomal markers) and low-density fractions
(mitochondrial markers).
mouse continuous liver After homogenization, lysosomes equilibrated in the dense regions of Percoll electron microscopy, Bradford protein assay, enzymatic assays 1104
gradients.
rat continuous cultured hepatocytes Lysosomal fractions were used to assay for endocytic transport of lysosomal purification of lysosomal membrane glycoprotein, Lowry protein 1105
membrane glycoprotein from cell surface to lysosomes. assay, protein-horseradish peroxidase assay
rat continuous liver Analysis of relevant marker enzymes showed considerably purified lysosomal Lowry protein assay, enzyme assays, free isoelectron focusing 24
(self-generating) particles in the density range of 1.04 to 1.11 g/mL.
rat, buffalo continuous (differential kidney The method gave a 25 to 40‑fold enrichment in lysosomal marker enzymes preparation of membrane vesicles, electron microscopy, protein assay 1106
and isopycnic) with < 0.5% contamination from mitochondrial and peroxisomal markers.
porcine continuous cultured kidney The method allowed for the relatively easy preparation of enriched fractions distribution and structure of vacuolar H+ ATPase, radiolabeling 1107
epithelial cells of endosomes and lysosomes. detection, hexosaminidase activity and alkaline phosphatase activity
55
Mitochondria
plant discontinuous etiolated tissue and green For etiolated tissue mitochondria, about 90% of catalase contamination cytochrome c oxidase (CCO) activity, membrane activity, respiratory 12
leaf tissue was removed. For green leaf mitochondria, about 95% of chlorophyll, control and substrate oxidation measurements
80% of catalase and 65% of glycollate oxidase were removed.
plant discontinuous (3-layer) etiolated tissue and green Separation of mitochondria from chloroplast material was possible under chlorophyll, cytochrome c oxidase and glycollate oxidase activities 43
leaf tissue isoosmotic conditions, and in a relatively short time.
rabbit, porcine discontinuous heart Percoll was especially suitable for in vitro studies on mitochondria from electron microscopy, enzyme activities 1108
both normal and diseased hearts.
rat discontinuous liver Isolated rat liver mitochondria were split into three density fractions when staining of mitochondrial populations, flow cytometry 1109
applied to a Percoll gradient.
Plasmo-dium continuous NA The purified mitochondria were obtained at the interface with a density mitochondrial marker enzyme assays, phase-contrast and electron 1110
berghei of 1.05 g/mL. microscopy
(protozoa)
turkey discontinuous (3-layer) sperm Mechanical disruption, sonication and centrifugation over Percoll was an fluorescence and electron microscopy, cytochrome oxidase assay, 1111
effective procedure to isolate the mitochondria. oxygen consumption, mitochondrial DNA isolation
56
Granules
human discontinuous whole blood neutrophils Specific and gelatinase granules were separated on a three-layer myeloperoxidase, alkaline phosphatase, lactoferrin, gelatinase, 1112
(3-layer) Percoll gradient. B12 binding protein, b2 micro- globulin, cytochrome b558, and
CD116 assays
human discontinuous whole blood neutrophils Subcellular fractionation resulted in a band containing gelatinase and specific receptor localization, enzyme marker assays 1113
(2-layer) granules and a band containing plasma membrane and secretory vessels.
human discontinuous whole blood neutrophils Percoll gradient centrifugation resulted in a bottom band containing azurophil marker enzyme assays, ELISA 1114
(2-layer) granules, a top band of plasma membrane and secretory vesicles, and a clear
super-natant containing cytosol.
human discontinuous whole blood neutrophils Percoll was used for subcellular fractionation of plasma membranes, specific subcellular localization of myeloperoxidase alkaline phosphatase, 1115
(2-layer) granules and azurophilic granules. and vitamin B12 binding protein
human continuous whole blood primary cultured Percoll gradients were used for the isolation of large granular lymphocyte (LGL) macrophage tumorcidal assay 1116
lymphocytes cytoplasmic granules.
mouse continuous mastocytoma mast cell Density gradient centrifugation was carried out in Percoll/0.25 M sucrose. uptake and degradation of mast cell granules by mouse peritoneal 17
macrophages
rat continuous parotid gland NA A secretory granular fraction (SG) and a plasma membrane-rich fraction (PM) enzyme assays, interactions of SG with PM 1117
were isolated using differential and Percoll gradient centrifugation.
bovine discontinuous adrenal gland NA Using Percoll to isolate chromaffin granules did not increase the yield, but electron microscopy, glutaraldelyde fixation for preparation of 78
(3-layer) it did eliminate the need for exposure of the granules to extreme hypertonic affinity column
conditions during isolation.
Paracentrotus discontinuous NA NA Lytic molecules were contained within small (0.1 to 0.25 mm) granules hemolytic and enzymatic activities 1118
lividus (sea urchin) (2-layer) (cytolytic granules) which could be isolated by Percoll gradients.
57
Plant organelles
Organelle Species Gradient type Tissue type Comments Downstream application Ref. #
mitochondria castor bean continuous seed (endosperm) Highly purified mitochondria were obtained with the Percoll gradient. mitochondrial cytidyl-transferase assay 1119
mitochondria sunflower continuous seed No organellar contamination was seen in the pellet sections. Lowry protein assay, characterization of NADP-dependent 1120
isocitrate dehydro-genase (NADP-IDH), SDS-PAGE and native gel
electrophoresis, gel filtration, electron microscopy
mitochondria maize, faba discontinuous leaf The purified intact mitochondria exhibited high respiratory controls in vitro radioactive labeling of the products of mitochondrial protein 1121
bean, wheat, and P/O ratios and were cleared of most of the chlorophyll. synthesis and their analysis by SDS-PAGE
tobacco,
sugar beet
chloroplast tobacco discontinuous leaf The yield from the Percoll gradient was 4.63 × 107 chloroplasts/g of Extraction of chloroplast proteins, Bradford protein assay, SDS-PAGE, 1122
(Nicotiana chlorophyll/chloroplast. protein blotting and immunological reactions
tabacum)
chloroplast Pea (Pisum continuous leaf The purified chloroplasts were capable of light-dependent protein synthesis in vitro reconstitution of protein transport and fractionation of 76
sativum) at rates comparable to those previously reported. chloroplast stromal protein
and spinach
(Spinacea
oleracea)
chloroplast spinach continuous linear leaf A clear separation of intact chloroplasts sustaining high photosynthetic enzyme assays, photosynthetic CO2 fixation, and O2 evolution 88
(Spinacea gradient activities occured.
oleracea)
cytoplasts various discontinuous leaf Cytoplasts were obtained by centrifugation of leaf protoplasts on Percoll cytoplast staining, laser microscopy, cytoplast: protoplast fusion 1123
gradients.
protoplasts barley discontinuous seed (aleurone After the Percoll gradient, the protoplasts were obtained in relatively high yield transient expression of CAT activity by transfected barley protoplasts 1124
(4-layer) layer) and showed good viability.
nuclei carrot discontinuous suspension cells This method yielded an average of 2 × 105 nuclei from 2 g of suspension cytochrome c oxidase and reductase assays, and in vitro RNA 1125
(3-layer) cultured cells (approximately 2 × 106 cells). Greater than 80% of the nuclei synthesis
appeared fully intact following the Percoll gradient.
plastids barley, pea, continuous leaf and seed Plastids obtained using Percoll exhibited high degrees of intactness starch synthesis, enzyme assays 1126
maize endosperm (89.1% and greater) and purity.
58
Miscellaneous organelles
nuclei chicken continuous skeletal muscle Percoll density gradient centrifugation provided a convenient method for the in vitro transcription 832
(self-generated) isolation of transcriptionally active nuclei applicable to a variety of tissues.
nuclei Neurospora continuous whole organism Percoll was a very effective alternative to LUDOX™ for the purification of electron microscopy, DNA, RNA and protein purification 1127
crassa (fungi) Neurospora nuclei from crude nuclear preparations.
nuclei and NA continuous cultured NIH and Percoll centrifugation allowed efficient fractionation and preservation of b-galactosidase and galactosyltransferase activity 597
sub-cellular (self-generated) KNIH cells enzymatic activity.
fractionation
endosomes human continuous cultured hepatoma Percoll gradients were used to separate endosomes from lysosomes. The b-hexosaminidase activity, Bradford protein assay 1130
cells conditions of centrifugation were chosen specifically to permit resolution
of early, intermediate and late endosomes.
endosomes human continuous cultured B cells Percoll was used to isolate intracellular major histocompatability complex sequence analysis of pooled and single peptides, fluorescence labeling 1131
(MHC) molecules in a preparative scale from endosomal compartments. and binding assay
plasma membrane, human continuous liver biopsy Percoll permited rapid analytical subcellular fractionation. Resolution of marker enzyme assays 13
endoplasmic organelles was good, and recoveries were high (86% to 105%).
reticulum,
lysosomes and
mitochondria
melanosomes, human continuous cultured Subcellular fractionation was used to determine the relationship between enzyme activity assays, immunoflourescence and immuno-electron 1132
lysosomes, melanocytes melanosomes, lysosomes and peroxisomes. microscopy
peroxisomes
azygospores Condiobolas discontinuous whole organism Recovery was 64% on average for a variety of soil types. microscopy 246
obscuros (fungi) isolated from soil
chromosomal and human continuous and cultured HeLa 53 Chromosomes were isolated free of cytoplasmic contamination. microscopy, Western blotting 677
mitotic clusters discontinuous and CHO cells
(self-generated)
peroxisomes rat continuous and liver enzyme assay, fatty acid oxidation studies 53
discontinuous
(self-generated)
59
Miscellaneous organelles (continued)
various human continuous blood Percoll-sucrose gradients were used to purify subcellular fractions to assay indirect immunocytofluorescence microscopy, ultrastructural 1133
(self-generated) for catalase. immunogold, enzyme activity assays
cytosol, NA continuous cultured Percoll gradients were used to separate subcellular organelles into various marker enzyme assays 1134
lysosomes, Golgi (self-generated) monoblastic cell fractions.
elements line
microbodies Cladosporium discontinuous whole organism Best results were obtained with a discontinuous Percoll gradient which yielded catalase and cytochrome oxidase assays 1135
resinae (fungi) a fraction enriched in microbodies and one enriched in mitochondria.
subcellular human continuous cultured HL-60 Percoll centrifugation allowed efficient fractionation and preservation of peroxidase, b-glucuronidase and acid phosphatase assays 727
fractionation (self-generated) cells enzymatic activity.
lipid vesicles Torpedo continuous electroplax tissue Intact vesicles were isolated. phosphate determination 392
californica (self-generated)
60
Appendix
Summary methodology charts
Scheme 1. Separation of cells on gradients of Percoll. Scheme 2. Separation of subcellular particles and some viruses on gradients of Percoll.
Prepare an osmotically-adjusted Stock Isotonic Percoll (SIP) which Make Stock Isotonic Percoll (SIP) by adding 1 volume of 2.5 M sucrose
is isotonic with cell preparation to 9 volumes of Percoll (page 10)
Dilute stock Percoll to lower density by addition of 0.25 M sucrose (page 11)
Dilute to desired starting densities with physiological saline solution (page 11)
Either Or
Mix preparation of particles with Percoll solution in 0.25 M sucrose
Preform the gradient by layering, gradient

former or by centrifugation (e.g., 30 000 × g Mix cell preparation with diluted iso-osmotic Centrifuge (e.g., 60 000 × g for 15 to 45 min. in an angle-head rotor) -
for 15 to 45 min. in angle-head rotor) Percoll to form a homogenous suspension Particles band on the self-generated gradients as it is formed (page 20)
(page 20)
Use Density Marker Beads to monitor the gradient (page 22)

Use Density Marker Beads to monitor the gradient (page 22)
Fractionate gradient by upward displacement (page 24)
Layer the cell suspension on preformed

gradient and centrifuge (e.g., 400 to 800 × g Centrifuge (e.g., 30 000 × g for
Analyze fractions for enzyme activities etc. (page 25)
for 10 to 20 min. in swinging bucket or 15 to 45 min. in an angle-head rotor)
angle-head rotor)
Either Or
Fractionate gradient by upward displacement (page 24) Use fraction directly for further experiments Remove Percoll by one of two techniques
Use cell fraction directly. Remove Percoll from cells by washing (page 26)
High speed centrifugation Gel filtration chromatography
Centrifuge at 100 000 × g in a swinging Sephacryl S-1000 Superfine will
bucket or angle-head rotor for 1.5 to 2 h. retard Percoll; collect biological
Downstream applications: Cell culture — Cytodex™ 1 and 3 QuickPrep mRNA Purification Biological material stays on top of the particles in the void volume
Kit (27-9254-01), QuickPrep Micro mRNA Purification Kit (27-9255-01) hard pellet of Percoll formed (page 26) (page 26)
61
References
Note: In portions of the list of references below, the numbering is not sequential. This is due to the way in which the list
was constructed. All references with numbers lower than 891 have been extracted from the original Percoll Reference
List (1992), and the numbering used in that List was maintained in this Manual. All references higher than 891 are new
and are sequential.
1. Parenchymal cell mass determinations in human parathyroid 9. Isolation of rat peritoneal mast cells by centrifugation on 16. Long term growth in vitro of human T cell blasts with
glands. Åkerström, G., Johansson, H. et al., Presented at Societé density gradients of Percoll. Enerbäck, L. and Svensson, I. maintenance of specificity and function. Kurnick, J.T., Grönvik,
International de Chirurgie, San Francisco (August 1979). J. Immunol. Methods. 39, 135–145 (1980). K.-O., Kimura, A.K. et al., J. Immunol 122, 1255–1260 (1979).
2. Density determinations of human parathyroid glands by density 10. Distribution of Ia-antigen-like molecules on non-lymphoid 17. Uptake and degradation of mast cell granules by mouse
gradients. Åkerström, G., Pertoft, H., Grimelius, L. et al., Acta tissues. Forsum, U., Klareskog, L. and Peterson, P.A. Scand. J. peritoneal macrophages. Lindahl, U., Pertoft, H. and Seljelid, R.
Path. Microbiol. Scand. Sect. A. 87, 91–96 (1979). Immunol. 9, 343–349 (1979). Biochem. J. 182, 189–193(1979).
3. Estimation of parathyroid parenchymal cell mass using density 11. Separation of human lymphocytes on the basis of volume and 18. Purification and steroidogenic responses of isolated rat luteal
gradients. Åkerström, G., Grimelius, L., Johansson, H. et al., Am. density. Hutchins, D. and Steel, C.M. In Separation of Cells and cells. McNamara, B.C., Booth, R. and Stansfield, D.A. Presented
J. Pathol. 99, 685–694 (1980). Subcellular Elements (Peeters, H., Ed.) Pergamon Press, Oxford at International Union of Biochemistry Meeting Toronto (1979).
and New York (1979). 19. Separation of lactotrophs from hyperplastic rat
5. Rapid isolation of pancreatic islets from collagenase digested
pancreas by sedimentation through Percoll at unit gravity. 12. Separation of chloroplasts from mitochondria utilising silica sol adenohypophysis using Percoll density gradients. Milligan, J.V.
Buitrago, A., Gylfe, E., Henriksson, C. et al., Biochem. Biophys. gradient centrifugation. Jackson, C., Dench, J.E., Halliwell, B. et Abstracts, Canad. Fed. Biol. Sci. Meeting, Vancouver (1979).
Res. Commun. 79, 823–828 (1977). al., Presented at The Wolffson Conference, University of Surrey 20. Isolation and characterization of hepatocytes and Küpffer cells.
(July 1978). Page, D.T. and Garvey, J.S. J. Immunol. Methods 27, 159–173
6. Isolation and characterization of cells from rat adipose tissue
developing into adipocytes. Björntorp, P., Karlsson, M., 13. Simple analytical subcellular fractionation of liver biopsies with (1979).
Pertoft, H. et al., J. Lipid Res. 19, 316–324 (1978). Percoll. Jenkins, W.J., Clarkson, E., Milson, J. et al., Clin. Sci. 57, 21. Isopycnic separation of cells and cell organelles by
29P (1979). centrifugation in modified colloidal silica gradients. Pertoft, H. and
7. Presence of alloreactive Ia antigens on murine intestine
epithelial cells. Curman, B., Kämpe, O., Rask, L. et al., 14. Coloured beads as density markers in isopycnic centrifugation. Laurent, T.C. In Methods of Cell Separation Vol. 1. (Catsimpoolas,
Scand. J. Immunol. 10, 11–15 (1979). Kågedal, L. and Pertoft, H. Abstracts, 12th FEBS Meeting, N., Ed.) Plenum Press, New York 25–65 (1977).
Dresden (1978). 22. Density gradients prepared from colloidal silica particles coated
8. Separation of lymphoid cells, mast cells and macrophages on
Percoll density gradient. Courtoy, R., Simar, L.J. and Delrez, M. 15. Separation of blood cells from healthy and leukemic donors polyvinylpyrrolidone (Percoll). Pertoft, H., Laurent, T.C., Låås, T.
Presented at a Meeting of the Belgian Society of Immunology. in Percoll gradients. Rogge, H., Christ, H., Grosshans, E. et al., et al., Anal. Biochem. 88, 271–282 (1978).
(December 1978). Abstracts, 12th FEBS Meeting, Dresden (1978). 23. The viability of cells grown or centrifuged in a new density
gradient medium, Percoll. Pertoft, H., Rubin, K., Kjellén, L. et al.,
Exp. Cell Res. 110, 449–457 (1977). 62
24. Heterogeneity of lysosomes originating from rat liver 32. Epithelial rat liver cells have cell surface receptors recognizing 41. Purification of human T and B cells by a discontinuous density
parenchymal cells. Metabolic relationship of subpopulations a phosphorylated carbohydrate on lysosomal enzymes. gradient of Percoll. Gutierrez, C., Bernabe, R.R., Vega, J. et al., J.
separated by density gradient centrifugation. Pertoft, H., Ullrich, K., Mersmann, G., Fleischer, M. et al., Hoppe-Seyler’s Z. Immunol. Methods 29, 57–63 (1979).
Wärmegård, B. and Höök, M. Biochem. J. 174, 309–317 (1978). Physiol. Chem. 359, 1591–1598 (1978). 42. Production of Ficoll, Percoll and albumin gradients by the
25. Characterization of rabbit sperm by equilibrium sedimentation 33. Specific binding of rat liver plasma membranes by rat liver cells. freeze-thaw method. Haff, L.A. Prep. Biochem. 9, 149–156 (1979).
in Percoll during frequent ejaculation. Oshio, S., Kaneko, S. and Öbrink, B., Wärmegård, B. and Pertoft, H. Biochem. Biophys. Res. 43. Isolation of intact higher-plant mitochondria. Jackson, C.
Mohri, H. Arch. Androl. 17, 189–194 (1986). Commun. 77, 665–670 (1977). and Moore, A.L. Plant Organelles, Methodological Surveys (B)
26. A two-step procedure for the purification of hepatitis B surface 34. Liberation of a fibrogenic factor from human blood monocytes, Biochemistr, Vol. 9, (Reid, E., Ed.) Ellis Horwood Ltd, Chichester,
antigen (HbsAg). Einarsson, M., Kaplan, L. and Pertoft, H. Vox ascites cells, cultured histiocytes and transformed mouse West Sussex, UK 1–12 (1979).
Sanguinis 41, 91–97 (1981). macrophages by treatment with SiO2. Aalto, M., Kulonen, E., 44. Human MLC activated suppressor cells — enrichment on
Rönnemaa, T. et al., Scand. J. Clin. Lab. Invest. 39, 205–214
27. The use of density gradients of Percoll for the separation of discontinuous density gradients. Kabelitz, D., Fink, U. and
(1979).
biological particles. Pertoft, H., Laurent, T.C., Seljelid, R. et al., In Reichert, A. Immunobiol. 156, 218 (1979).
Separation of Cells and Subcellular Elements, (Peeters, H., Ed.) 35. Parenchymal cell mass determinations in human parathyroid 45. Physical chemical characterization of Percoll. I. Particle weight
Pergamon Press Oxford and New York 67–72 (1979). glands and its application in a material of hyperparathyroidism. of the colloid. Laurent, T.C., Pertoft, H. and Nordli, O. J. Colloid
Åkerström, G., Grimelius, L., Johansson, H. et al., World. J. Surg.
28. Collection of dinoflagellates and other marine microalgae by Interface Sci. 76, 124–132 (1980).
5, 555–563 (1981).
centrifugation in density gradients of a modified silica sol. 46. Physical chemical characterization of Percoll. II. Size and
Price, C.A., Reardon, E.M. and Guillard, R.R.L. Limnol. Oceangr. 36. In vitro induction of self reactive T lymphocyte memory in interaction of colloidal particles. Laurent, T.C., Ogston, AJ.G.,
123, 548–553 (1978). cultures of syngeneic peanut agglutinin-negative mouse Pertoft, H. et al., J. Colloid Interface Sci. 76, 133–141 (1980).
thymocytes and spleen cells. Born, W. and Wekerle, H.
29. Adhesion of rat hepatocytes to collagen. Rubin, K., Oldberg, Å.,
Immunobiol. 156, 243–244 (1979). 47. Physical chemical characterization of Percoll. III. Sodium
Höök, M. et al., Exp. Cell Res. 117, 165–177 (1978). binding. Laurent, T.C. and Pertoft, H. J. Colloidal Interface Sci.
37. Characteristics of cultured brain capillaires. Bowman, P.D.,
30. Macrophage functional heterogeneity: evidence for different 76, 142–145 (1980).
Betz, A.L. and Goldstein, G.W. J. Cell Biol. 83, 95a (1979).
antibody-dependent effector cell activities and expression of 48. Peanut agglutinin. IV. A tool for studying human mononuclear
Fc-receptors among macrophage subpopulations. Serio, C., 38. Characterization of bone marrow osteoprogenitor cell lines. cell differentiation. London, J., Perrot, J.Y., Berrih, S. et al.,
Gandour, D.M and Walker, W.S. J. Reticuloendothelial Soc. 25, Budenz, R.W. and Bernard, G.W. J. Cell Biol. 83, 32a (1979). Scand. J. Immunol. 9, 451–459 (1979).
197–216 (1979). 39. Isolation, characterization and cultivation of human 49. Conversion of 4-hydroxyphenylpyruvic acid into homogentisic
31. Rapid isolation of mouse Leydig cells by centrifugation trophoblastic cells. Calaminus, J.M., Brüggen, J. and Sorg, C. acid at the thylakoid membrane of Lemna gibba. Löffelhardt, W.
in Percoll density gradients with complete retention of Immunobiol. 156, 287 (1979). and Kindl, H. FEBS LETT. 104, 332–334 (1979).
morphological and biochemical integrity. Schumacher, M., 40. Characterization of procoagulant activity produced by cultures
Schäfer, G., Holstein, A.F. et al., FEBS LETT. 91, 333–338 (1978). 50. Further purification of rat spermatogenic cells by density
of human monocytes and lymphocytes separated in colloidal centrifugation. Meistrich, M.L., Longtin, J.L. and Brock, W.A. J.
silica-polyvinylpyrrolidone gradients. Giddings, J.C., Piovella, F., Cell Biol. 83, 226a (1979).
Ricetti, M. et al., Clin. Lab. Haematol. 2, 121–128 (1980).
63
51. Protozoan parasite-induced proliferative response of primed T 60. Harvesting of marine microalgae by centrifugation in density 68. Recognition of human urine ~-N-acetylglucosaminidase by rat
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73
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Ordering information
Products for cell separation and culture Products for purification of RNA (A) (B) (C)
Product Quantity Code number Production Quantity Code number
Percoll 1l 17-0891-01 QuickPrep Micro mRNA Purification Kit 1 kit† 27-9255-01

(24 purifications)
250 mL 17-0891-02
QuickPrep mRNA Purification Kit 1 kit† 27-9254-01
Percoll PLUS 1l 17-5445-01 (4 purifications)
250 mL 17-5445-02 mRNA Purification Kit
Ficoll-Paque PLUS 6 × 100 mL 17-1440-02 (2 purifications) 1 kit† 27-9258-01
6 × 500 mL 17-1440-03 (4 purifications) 1 kit† 27-9258-02
Ficoll-Paque PREMIUM 6 × 100 mL 17-5442-02 CsTFA (Solution) 100 mL 17-0847-02
6 × 500 mL 17-5442-03 Oligo(dT)-Cellulose Type 7 1g 27-5543-02
Ficoll PM 70 100 g 17-0310-10
500 g 17-0310-50 Kits for cDNA synthesis

5 kg 17-0310-05 Product Quantity Code number
Ficoll PM 400 100 g 17-0300-10 TimeSaver cDNA Synthesis Kit (5 reactions) 1 kit† 27-9262-01
Panel (A): Separation of human blood cells in a gradient of Percoll. Bottom layer
500 g 17-0300-50 First-Strand cDNA Synthesis Kit (55 reactions) 1 kit †
27-9261-01 contains red blood cells, the middle band is polymorphonuclear cells
5 kg 17-0300-05 Ready-To-Go™ T-Primed First-Strand Kit (50 reactions) 1 kit† 27-9263-01 (e.g., granulocytes) and the top band is mononuclear cells.
40 kg 17-0300-08 Panel (B): Percoll, our exceptional density gradient medium, is available in
†
Product must be shipped cold. There is an extra charge for insulated container and refrigerant.
easy to open, resealable bottles.
Cytodex 1 25 g 17-0448-01
Panel (C): Percoll PLUS reagent separates various cell types for clinical research
Cytodex 3 10 g 17-0485-01 applications and is available in easy to open resealable 250 mL and 1 l bottles.
76
cytiva.com/cellprep
Cytiva and the Drop logo are trademarks of Global Life Sciences IP Holdco LLC or
an affiliate. Percoll, Sephadex, Sephacyl, Sepharose, Ficoll, Ficoll-Paque, Cytodex, and
Ready-To-Go are trademarks of Global Life Sciences Solutions USA LLC or an affiliate
doing business as Cytiva.
Percoll PLUS is protected by the following patents and equivalent patents and
patent applications in other countries, which are licensed to Cytiva from Dendreon
Corporation: US patent number 4,927,749, US patent number 4,927,750, Canadian
patent number 1,338,492, Japanese patent number 2,628,509, US patent number
5,789,148, US patent number 6,015,843 and European patent number 1,047,635. A free,
non-transferable license to use this product for density gradient separation purposes
under the above mentioned patent rights accompanies the purchase of the product
from a Cytiva company and its licensed distributors, but any use of Percoll PLUS or any
other organosilanized colloidal silica particle-based separation media to enrich, purge
or isolate cells for active immunotherapy for oncology applications shall be excluded
from such license.
Beckman is a trademark of Beckman Coulter, Inc. Coomassie is a trademark of Thermo
Fisher Scientific, Ltd. LUDOX is a trademark of W.R. Grace & Co.-Conn. Merthiolate is
a trademark of Eli Lilly and Company Triton is a trademark of Union Carbide Chemicals
and Plastic Company Inc. All other third-party trademarks are the property of their
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Methodology and applications

Separation by size (rate zonal centrifugation) Percoll

Buoyant density g/mL

Particle size composition

(ro- ri) Voro + Vxr10

ry = density of diluting medium in g/mL

(density of 0.25 M sucrose is ~1.032 g/mL)*

assuming: 2880 = osmolality of 1.5 M NaCl

V = Volume of final working solution (mL)

r = desired density of final working solution (g/mL)

ro = density of Percoll undiluted (g/mL)

1.07 - 1/11 × 1.058 - (1-1/11)

Effects of osmolality on apparent buoyant density of cells and 1.15

Number of cells × 106

Enzyme activity % of total

10 8.5 310 1.091

20 8.5 374 1.110 40

30 8.5 503 1.148 30

Distance from the meniscus cm

1.00 1.10 1.20 Density g/mL

the gradient during fractionation. 90%

Density of Percoll solution g/mL

Gradients formed in situ

3. Add 10 µl of a suspension of each type of Density Marker Beads to each

Density Marker Bead — properties

Density of each bead type: calibrated to ± 0.0005 g/mL

Buoyant density g/mL

Buoyant density g/mL

Protein determination and enzyme assay

Washing (low speed centrifugation)

Washing (high speed centrifugation) permission of the authors and publisher).

(Original work by Pertoft et al. reproduced by kind permission)

Sterilization of Percoll solutions

Aggregates of silica particles

Preform the gradient by layering, gradient

Use Density Marker Beads to monitor the gradient (page 22)

Fractionate gradient by upward displacement (page 24)

Layer the cell suspension on preformed

Percoll 1l 17-0891-01 QuickPrep Micro mRNA Purification Kit 1 kit† 27-9255-01

500 g 17-0310-50 Kits for cDNA synthesis

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3. Add 10 µl of a suspension of each type of Density Marker Beads to each