Biofiles

Volume 6, Number 5

Centrifugation
Centrifugation Basics
Density Gradient Media
Cell Viability and Proliferation
Organelle Isolation
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Centrifugation Basics 4
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Highlights from this issue: ECACC® Cell Lines 26

Centrifugation is one of the most basic
of laboratory applications and is used Centrifugation Equipment 27
by a wide range of clinical and research
personnel. However, information on
centrifugation theory and separation Cover: The ferris wheel is an amusement
techniques are usually only found in park ride making use of centrifugal force
centrifuge instrument manuals or by contacting the manufacturers
and the Earth's gravitational field. Riders
of density gradient media directly. This issue of Biofiles is intended to
feel "heaviest" at the bottom of the axis
provide a basic understanding of how biological particles behave in
of rotation and "lightest" at the top.
a centrifugal field and the types of density gradient media used to
separate them.

Coming Next Issue:

The next issue of Biofiles will focus on the
challenging problem of cell culture
contamination. Cell cultures are
vulnerable to a wide variety of
contaminants including:

• Chemical
• Viral
• Bacterial, including mycoplasma
• Fungal
• Insects
• Cross contamination with other cell lines
This issue will address what Sigma
can offer to combat cell culture contamination.

Technical content: Mark Frei

Technical Marketing Specialist
[email protected]
Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 3

Introduction
Mark Frei
Technical Marketing Specialist
[email protected]

Separation of particles by sedimentation In 1977, the stabilized silica colloid coated

is one of the most powerful tools in with polyvinylpyrrolidone (PVP) called
biology. Even though sedimentation using Percoll® became available for separating
centrifugation is not a new technology, it cells and subcellular particles. In 1968,
is essential for cutting edge genomic and Boyum described methods for the isolation
proteomic research by providing purified of mononuclear cells from circulating
particles of interest. In a survey at the US blood and bone marrow using mixtures of
National Institutes of Health, over 65% polysaccharide and a radiopaque contrast
of research workers replied that using medium. This led to the development of
centrifugation to purify cells, subcellular the first nonionic iodinated density gradient
organelles, viruses, proteins, and nucleic medium, metrizamide, in the 1970s.3 Now,
acids is an integral part of their work.1 a large selection of commercial iodinated
Density gradient centrifugation is a density gradient media are available.
technique that allows the separation of This issue of Biofiles examines the theory
particles on the basis of their size, shape, and practice of biological separation. The
and density. A density gradient is typically beginning section provides a primer on
created by layering media of increasing the basic concepts of centrifugation. Three
density in a centrifuge tube. When a sample basic types of centrifugal separations are
is layered on top of a density gradient and highlighted; differential centrifugation,
centrifuged, the various particles move rate-zonal centrifugation, and isopycnic
through the gradient at different rates. The centrifugation. A concise description of each
particles appear as bands or zones in the is given along with the separation principles
gradient with the more dense and larger involved. Cell viability kits, technical support
particles migrating furthest. information, and centrifugation equipment
A number of different compounds have are also included.
been investigated as density gradient We hope you find the reference information
media. One of the first density gradient and products relevant. For a comprehensive
centrifugation techniques was developed list of our centrifugation media products,
in the 1950s and used a buffered sucrose please visit sigma.com/handh
solution for the purification of cell organelles.
Sucrose quickly became the density medium References:
of choice for separating homogenized 1. Biological Centrifugation, J. Graham, p. 1
2. Centrifugation, A Practical Approach (2nd Edition), D.
mammalian tissues. Later, cesium chloride Rickwood, p.35
gradients were used to separate DNA of 3. Iodinated Density Gradient Media, A Practical Approach, D.
Rickwood, p.1
different densities. Meselson and Stahl in
1958 used cesium chloride density gradient
centrifugation in an elegant experiment to
support the semi-conservative model of DNA
replication. Colloidal-silica suspensions were
first manufactured by DuPont and sold under
the name of LUDOX®.2
4

Centrifugation Basics

Centrifugation Basics
The earth's gravitational force is sufficient to Most particles are so small that gravitational
separate many types of particles over time. d 2 (p–L) 3 g force is insufficient to overcome the random
v=
A tube of anticoagulated whole blood left 18 n molecular forces of particles to influence
standing on a bench top will eventually v = sedimentation rate or velocity of the sphere
separation. Centrifugation, the name given
separate into plasma, red blood cell and white d = diameter of the sphere to separation applications which involve
blood cell fractions. However, the length p = particle density spinning around an axis to produce a
L = medium density
of time required precludes this manner of n = viscosity of medium
centrifugal force, is a way to increase the
separation for most applications. In practice, g = gravitational force magnitude of the gravitational field. The
centrifugal force is necessary to separate most particles in suspension experience a radial
particles. In addition, the potential degradation Figure 1. The Stokes equation. centrifugal force moving them away from the
of biological compounds during prolonged axis of rotation.2 The radial force generated
From the Stokes equation five important
storage means faster separation techniques by the spinning rotor is expressed relative to
behaviors of particles can be explained:
are needed. the earth's gravitational force and therefore is
1. The rate of particle sedimentation is known as the relative centrifugal force (RCF)
The rate of separation in a suspension of
proportional to the particle size. or the "g force." The g force acting on particles
particles by way of gravitational force mainly
depends on the particle size and density. 2. The sedimentation rate is proportional is exponential to the speed of rotation
Particles of higher density or larger size to the difference in density between the (defined as revolutions per minute; rpm).
typically travel at a faster rate and at some particle and the medium. Doubling the speed of rotation increases
point will be separated from particles less the centrifugal force by a factor of four. The
3. The sedimentation rate is zero when the
dense or smaller. This sedimentation of centrifugal force also increases with the
particle density is the same as the medium
particles, including cells, can be explained distance from the axis of rotation. These two
density.
by the Stokes equation, which describes parameters are of considerable significance
4. The sedimentation rate decreases as the when selecting the appropriate centrifuge.
the movement of a sphere in a gravitational
medium viscosity increases. Table 1 summarizes the applications that can
field.1 The equation calculates the velocity of
sedimentation utilizing five parameters (see 5. The sedimentation rate increases as the be classified by the relative centrifugal force.3
Figure 1). gravitational force increases.

Parameters Low speed High speed Ultracentrifuge

Speed ranges (r.p.m. x 103) 2–6 18–22 35–120
Maximum RCF (x 103) 8 60 700
Pelleting Applications
Bacteria - Yes Yes*
Animal and plant cells Yes Yes Yes*
Nuclei Yes Yes Yes*
Precipitates Some Most Yes*
Membrane organelles Some Yes Yes
Membrane fractions Some Some Yes
Ribosomes/polysomes - - Yes
Macromolecules - - Yes
Viruses - Most Yes
* Can be done but not usually used for this purpose

Table 1. Classes of centrifuges and their applications

 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 5

RCF is dependent on the speed of rotation Radius

(cm) g Rpm
Nomogram instructions:
30 5000
in rpm and the distance of the particles from 7000
6000 4500 1. Measure the radius (cm) from the center
5000
the center of rotation. When the speed of 25
4000 4000
of the centrifuge rotor to the end of test
3000 3500
rotation is given in rpm (Q) and the distance 20
tube carrier.
19 2000
18 3000
(r) is expressed in centimeters, RCF can be 17 1500
16
calculated by using the formula in Figure 2. 15 1000 2500 2. Obtain the relative centrifugal force
14 800
13 700
600 necessary for the application.
12 500 2000
11 400
2 3. A straight line connecting the value of the
Q 10 300
RCF = 11.18 3 r 3 9 200
1500
radius with the relative centrifugal force
1000 8 150
1400
1300

7 100
1200 (g) value will enable the speed of the rotor
Figure 2. Formula for relative centrifugal force (RCF) 80 1100
6
70
60
1000 (rpm) to be read off of the right column.
50 900
A nomogram can also be used to obtain 5 40
800
the speed of a centrifuge rotor necessary 30 References:
700
4 20
for a desired RCF (see Figure 3). This 15
1. Laboratory Techniques in Biochemistry and Molecular
600
Biology, P.T. Sharpe, p.18
10
quick estimate is useful for low speed 3 500 2. Biological Centrifugation, J. Graham, p. 3
A B C 3. Centrifugation, Essential Data, D. Rickwood, p.12
centrifugation applications. However, it is
more accurate to use the RCF calculation for
speeds in excess of 10,000 rpm. Figure 3. Nomogram for estimation of centrifuge rpm
setting.

biomolecules

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6

Centrifugation Separations

Centrifugation Separations
There are two types of centrifugal Differential pelleting is commonly used Sample zone
techniques for separating particles, A. B. C.
for harvesting cells or producing crude
differential centrifugation and density subcellular fractions from tissue homogenate.
gradient centrifugation. Density gradient For example, a rat liver homogenate

Centrifugal Force
centrifugation can further be divided into containing nuclei, mitochondria, lysosomes,
rate-zonal and isopycnic centrifugation. and membrane vesicles that is centrifuged Density
gradient
at low speed for a short time will pellet
Differential Centrifugation mainly the larger and more dense nuclei.
The simplest form of separation by Subsequent centrifugation at a higher
centrifugation is differential centrifugation, centrifugal force will pellet particles of the
sometimes called differential pelleting (see next lower order of size (e.g., mitochondria) Figure 2. Rate-Zonal Centrifugation
Figure 1). Particles of different densities and so on. It is unusual to use more than four
Sample is layered as a narrow zone on the top of a density
or sizes in a suspension will sediment differential centrifugation cycles for a normal gradient (2B). Under centrifugal force, particles move at
tissue homogenate. different rates depending on their mass (2C).
at different rates, with the larger and
denser particles sedimenting faster. These Due to the heterogeneity in biological The speed at which particles sediment
sedimentation rates can be increased by particles, differential centrifugation suffers depends primarily on their size and mass
using centrifugal force. A suspension of from contamination and poor recoveries. instead of density. As the particles in the
cells subjected to a series of increasing Contamination by different particle types can band move down through the density
centrifugal force cycles will yield a series be addressed by resuspension and repeating medium, zones containing particles of similar
of pellets containing cells of decreasing the centrifugation steps (i.e., washing the size form as the faster sedimenting particles
sedimentation rate. pellet).1 move ahead of the slower ones. Because the
density of the particles is greater than the
A. B. C. D.
Rate-Zonal Centrifugation density of the gradient, all the particles will
In rate-zonal centrifugation the problem of eventually form a pellet if centrifuged long
enough.2
Centrifugal Force

cross-contamination of particles of different

sedimentation rates may be avoided by
layering the sample as a narrow zone on Isopycnic Centrifugation
top of a density gradient (see Figure 2). In In isopycnic separation, also called buoyant
this way the faster sedimenting particles are or equilibrium separation, particles are
not contaminated by the slower particles separated solely on the basis of their density.
Centrifugation Time as occurs in differential centrifugation. Particle size only affects the rate at which
However, the narrow load zone limits the particles move until their density is the same
Figure 1. Differential Centrifugation volume of sample (typically 10%) that can be as the surrounding gradient medium. The
Particles of different densities or size will sediment at accommodated on the density gradient. The density of the gradient medium must be
different rates with the largest and most dense particles
sedimenting the fastest followed by less dense and
gradient stabilizes the bands and provides a greater than the density of the particles to
smaller particles. medium of increasing density and viscosity. be separated. By this method, the particles
will never sediment to the bottom of the
tube, no matter how long the centrifugation
time (see Figure 3).
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 7

A. B.
Suitable Density Gradient Medium No single compound can satisfy all of the
Selection above criteria. Therefore a wide range of
gradient media are used for the different types
The primary function of density gradient
Centrifugal Force

of samples (see Table 1). Most media are

centrifugation is to separate particles, either on
capable of producing the range of densities
the basis of their buoyancy density or their rate
required and being easily removed from the
of sedimentation. For rate-zonal separations,
particles of interest.
the function of the gradient is to provide a
gradient of viscosity which improves particle The effect of osmolality on biological particles
resolution while stabilizing the column from requires special consideration. The osmolality
convection currents. For isopycnic separations, of most mammalian fluids is 290-300 mOsm.4
Figure 3. Isopycnic Centrifugation
the important feature is that the maximum This is the osmolality of balanced salt solutions
Starting with a uniform mixture of sample and density (e.g., 0.85-0.9% NaCl) and most common
gradient (3A) under centrifugal force, particles move until density of the gradient media is higher than
their density is the same as the surrounding medium (3B). that of the particles. An ideal density gradient media. High osmolality solutions not only
media has the following properties:3 remove water from the interior of membrane-
Upon centrifugation, particles of a specific bound particles, they also remove water
density sediment until they reach the point • Sufficient solublilty to produce the range of bound to macromolecules like DNA. Loss
where their density is the same as the gradient densities required
of water from cells will reduce their size and
media (i.e., the equilibrium position). The • Does not form solutions of high viscosity in increase their density, thereby affecting their
the desired density range
gradient is then said to be isopycnic and the
particles are separated according to their
• Is not hyperosmotic or hypoosmotic when buoyancy and rate of sedimentation. The
the particles to be separated are osmotically osmotic effect on cells and macromolecules
buoyancy. Since the density of biological sensitive may be reversible, though it is a possible
particles is sensitive to the osmotic pressure • Solutions of the gradient should be source of error that should be avoided.
of the gradient, isopycnic separation may adjustable to the pH and the ionic strengths
vary significantly depending on the gradient Over the years, a variety of different
that are compatible with the particles being
medium used. Although a continuous separated compounds have been developed as
gradient may be more suited for analytical • Does not affect the biological activity of the density gradient media in order to enhance
purposes, preparative techniques commonly sample the separation process and to overcome
use a discontinuous gradient in which the • Nontoxic and not metabolized by cells osmolality and viscosity problems. There are
particles band at the interface between the • Does not interfere with assay procedures or five main classes of density gradient medium:
react with the centrifuge tubes
density gradient layers. This makes harvesting
• Exhibits a property that can be used as a
• Polyhydric (sugar) alcohols
certain biological particles (e.g., lymphocytes)
measure of concentration
• Polysaccharides
easier.
• Easily removed from the purified product
• Inorganic salts
• Autoclavable
• Iodinated compounds
• Reasonable cost
• Colloidal silica
Gradient medium type Principle uses
Polyhydric alcohols
Sucrose Organelles, membrane vesicles, viruses, proteins, ribosomes, polysomes
Glycerol Mammalian cells (infrequent), proteins
Sorbitol Nonmammalian subcellular particles
Polysaccharides
Ficoll®, polysucrose and dextrans Mammalian cells (sometimes in combination with iodinated density gradient media), mammalian subcellular particles
(infrequent)
Inorganic salts
CsCl DNA, viruses, proteins
Cs2SO4 DNA, RNA
KBr Plasma lipoproteins
Iodinated gradient media
Diatrizoate Mainly as a component of commercial lymphocyte isolation media
Nycodenz®, Histodenz™ Mammalian cells, organelles, membrane vesicles, viruses
Iodixanol Mammalian cells, organelles, membrane vesicles, viruses, plasma lipoproteins, proteins, DNA
Colloidal silica media
Percoll® Mammalian cells, organelles, membrane vesicles (infrequent)
Table 1. Density gradient media types and their principle uses.
8

Polyhydric alcohols  for molecular biology, ≥99.5% (GC) phosphatases................................................................... none detected
DNase, RNase, protease..............................none detected proteases............................................................................. none
Polyhydric alcohols are considered Free glucose..............................<0.1% detected
nonionic gradient media. Some of the first heavy metals (as Pb).............................. <5 ppm RNases.................................................................................. none detected
centrifugation techniques developed in S0389-500G 500 g ign. residue.................... ≤0.1% Fe............................... ≤1 mg/kg
the 1950s used sucrose in the purification S0389-1KG 1 kg pH.......5.5-8, 5 M H2O (25 °C) K.............................. ≤20 mg/kg
S0389-5KG 5 kg chloride (Cl-)......... ≤1 mg/kg Li................................ ≤1 mg/kg
of cell organelles. Sucrose gradients are
sulfate (SO42-)..... ≤10 mg/kg Mg............................ ≤1 mg/kg
widely used for the rate-zonal separation  BioReagent, suitable for cell culture, suitable for Ag................................ ≤5 mg/kg Mn............................ ≤1 mg/kg
of macromolecules and for the isopycnic insect cell culture, ≥99.5% (GC) Al................................. ≤1 mg/kg Mo............................ ≤1 mg/kg
separation of viruses and cell organelles. Use to create sucrose gradients for purification of As............................. ≤0.1 mg/kg Na........................... ≤20 mg/kg
viruses and proteins. Ba................................ ≤1 mg/kg NH4+......................... ≤5 mg/kg
The advantages are its stable nature, Bi.................................. ≤1 mg/kg Ni............................... ≤1 mg/kg
S1888-500G 500 g
inertness, and low cost. The disadvantages Ca................................ ≤5 mg/kg Pb.............................. ≤1 mg/kg
S1888-1KG 1 kg
lie in the fact that concentrated solutions S1888-5KG 5 kg
Cd................................ ≤1 mg/kg Sr................................ ≤1 mg/kg
are viscous and hypertonic. Reagent-grade Co................................ ≤1 mg/kg Tl................................ ≤5 mg/kg
Cr................................. ≤1 mg/kg Zn............................. ≤1 mg/kg
sucrose may be contaminated with RNAses Glycerol
Cu............................... ≤1 mg/kg
or heavy metals and therefore are unsuitable 1,2,3-Propanetriol; Glycerin
[56-81-5] HOCH2CH(OH)CH2OH FW 92.09 Lit cited: 1. R.C. Ogden, D.A. Adam, Meth. Enzymol. 152, 79 (1987); 2.
for DNA and RNA purification. Glycerol H. Miller, Meth. Enzymol. 152, 145 (1987);
density.............................................................................................1.25 g/mL
solutions are less dense than corresponding 49767-100ML 100 mL
 for molecular biology, ≥99%
sucrose solutions. However, glycerol 49767-250ML 250 mL
reagent 49767-1L 1 L
solutions of the same density of sucrose
DNase, RNase, NICKase, and protease...............none
solutions are much more viscous. Glycerol
helps to preserve the activity of certain
detected
Polysaccharides
Fe.............................. ≤5 ppm Mg.............................. ≤5 ppm
enzymes and it can be removed through heavy metals (as Pb)................................................................. <5 ppm
Polysaccharides circumvent the high
vacuum. G5516-100ML 100 mL
osmotic strength issues that arise with
G5516-500ML 500 mL using sucrose solutions. The most common
Sucrose G5516-1L 1 L polysaccharide medium used is Ficoll®. Ficoll
α-D-Glucopyranosyl β-D-fructofuranoside; α-D- is produced by the polymerization of sucrose
 BioXtra, ≥99% (GC)
Glc-(1→2)-β-D-Fru; D(+)-Saccharose; Sugar; β-D-Fructo-
furanosyl-α-D-glucopyranoside Phosphorus (P)...........................................................................≤0.0005% molecules with epichlorohydrin to give a
[57-50-1] C12H22O11 FW 342.30 ign. residue.................... ≤0.1% K.................................... ≤0.005% polysaccharide with the average molecular
chloride (Cl-)............. ≤0.001% Mg.............................. ≤0.0005% weight of 400,000. Ficoll solutions below
 BioXtra, ≥99.5% (GC)
sulfate (SO42-)........... ≤0.002% Na.................................. ≤0.005%
Insoluble matter.............................. passes filter test 20% (w/v) have a density of 1.07 g/cm3 and
Al................................ ≤0.0005% NH4+................................ ≤0.05%
ign. residue............. ≤0.01% Cu..................... ≤0.0005%
Ca................................ ≤0.0005% Pb................................... ≤0.001% are considered osmotically inert. The main
(as SO4) Fe.............................. ≤0.0005% disadvantage is Ficoll solutions are more
Cu................................ ≤0.0005% Zn............................... ≤0.0005%
pH........5.5-7.5, 1 M H2O (20 °C) K.............................. ≤0.005%
Fe................................ ≤0.0005% viscous than comparable sucrose solutions.
chloride (Cl-)..................≤0.005% Li.............................. ≤0.0005%
sulfate (SO42-)............≤0.005% Mg..............................≤0.0005% G6279-500ML 500 mL
Al.............................. ≤0.0005% Mn............................. ≤0.0005% G6279-1L 1 L Dextran solution from Leuconostoc
As.............................. ≤0.0001% Mo............................. ≤0.0005% G6279-4X4L 4 × 4 L mesenteroides
Ba.............................. ≤0.0005% Na.............................. ≤0.005% [9004-54-0]
 ≥99% (GC)
Bi.............................. ≤0.0005% Ni.............................. ≤0.0005% Use of dextrans as long and hydrophilic spacer
heavy metals (as Pb).............................. ≤5 ppm
Ca.............................. ≤0.001% Pb.............................. ≤0.0005% arms to improve the performance of immobilized
Cd.............................. ≤0.0005% Sr.............................. ≤0.0005% G9012-100ML 100 mL proteins acting on macromolecules.
Co.............................. ≤0.0005% Zn.............................. ≤0.0005% G9012-500ML 500 mL
Cr.............................. ≤0.0005% G9012-1L 1 L  20 % (w/w) (Autoclaved)
G9012-2L 2 L average mol wt ~500,000
S7903-250G 250 g
G9012-1GA 1 gal store at:: 2–8 °C
S7903-1KG 1 kg
S7903-5KG 5 kg  BioUltra, for molecular biology, anhydrous, D8802-25ML 25 mL
≥99.5% (GC) D8802-50ML 50 mL
 ≥99.5% (GC)
Component of loading buffer in agarose gel
RNase..............................none detected
electrophoresis of nucleic acids previously
S9378-10MG 10 mg denatured with glyoxal1; Preparation of phage and
S9378-500G 500 g plasmid DNA, for the storage of pure cultures2
S9378-1KG 1 kg DNases.................................................................................. none
S9378-5KG 5 kg
detected
S9378-10KG 10 kg
insoluble matter........................................................... passes filter
test
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 9

Dextran sulfate sodium salt from Ficoll® 400 Colloidal Silica Media

Leuconostoc spp.  lyophilized powder, γ-irradiated, BioXtra, suitable
for cell culture
Colloidal silica media are not true solutions,
[9011-18-1]
Dialyzed but are colloidal suspensions of silica
 mol wt 6,500-10,000
F8636-25G 25 g
particles coated with polyvinylpyrrolidone
D4911-1G 1 g
D4911-10G 10 g
(PVP) with a diameter of 15–30 nm. The most
D4911-50G 50 g Ficoll® 400 widely known colloidal silica medium is
D4911-100G 100 g  lyophilized powder, Type 400-DL, BioReagent, Percoll®. The polyvinylpyrrolidone minimizes
suitable for cell culture
 average mol wt 9,000-20,000 the particle interactions with biological
Dialyzed
D6924-1G 1 g material and stabilizes the colloid. Being
F8016-5G 5 g
D6924-10G 10 g a colloid, the osmotic strength of Percoll
F8016-100G 100 g
D6924-50G 50 g
F8016-500G 500 g
is extremely low and changes little with
 average mol wt >500,000 (dextran starting density. The osmolality of Percoll gradients
material), contains 0.5-2.0% phosphate buffer, Ficoll® 400 can be adjusted by adding appropriate
pH 6-8  Type 400-DL, lyophilized powder amounts of sucrose or buffer solution. Percoll
store at:: 2–8 °C
Dialyzed gradients self-form when centrifuged in
D6001-1G 1 g
D6001-10G 10 g
F9378-5G 5 g fix-angle rotors (swing out rotors are not
F9378-10G 10 g satisfactory for self-forming gradients).5
D6001-50G 50 g
F9378-25G 25 g
D6001-100G 100 g Percoll can be removed from suspensions
F9378-100G 100 g
D6001-500G 500 g by differential pelleting. Percoll gradients
F9378-500G 500 g
Precipitating agent in the quantitation of HDL are mainly used in isopycnic separations of
cholesterol Ficoll® 400 ▲
cells, organelles, membrane vesicles, and
D8787-1G 1 g even some viruses. The main limitation is
D8787-5G 5 g Ficoll® PM 70
Poly(sucrose-co-epichlorhydrin) that the sample particle size must be larger
Ficoll® solution [72146-89-5] than the colloidal silica particles, otherwise
[26873-85-8] A nonionic synthetic polymer of sucrose. the particles of silica pellet before the sample
A nonionic synthetic polymer of sucrose.  Type 70 bands.
Used in electrophoresis and as a hapten carrier. mol wt ~70,000
Most commonly used to prepare density gradients. Percoll®
F2878-100G 100 g
 Type 400, 20% in H2O F2878-500G 500 g Percoll® consists of colloidal silica particles of
15–30 nm diameter (23% w/w in water) which
0.2 μm filtered
Ficoll® PM 400 have been coated with polyvinylpyrrolidone (PVP).
store at: 2–8 °C
Polysucrose 400 [26873-85-8]
It is used in cell separation and organelle isolation.
F5415-25ML 25 mL
A nonionic synthetic polymer of sucrose. aseptically filled
F5415-50ML 50 mL
Used for cell separation and organelle isolation.  pH 8.5-9.5 (25 °C)
store at: 2–8 °C
▼ Ficoll® 400  Type 400
Polysucrose 400 [26873-85-8] P1644-25ML 25 mL
powder
A nonionic synthetic polymer of sucrose. P1644-100ML 100 mL
spray-dried P1644-500ML 500 mL
Used for cell separation and organelle isolation.
F4375-10G 10 g P1644-1L 1L
Ficoll® 400 F4375-25G 25 g
F4375-100G 100 g  pH 8.5-9.5 (25 °C), cell culture tested
 BioXtra, Type 400-DL, lyophilized powder density..................................................1.13 g/mL±0.005 g/mL, 25 °C
Insoluble matter................................................................................≤0.1% F4375-500G 500 g
store at: 2–8 °C
Phosphorus (P)............................................................................≤0.0005%
Polysucrose 400 P4937-25ML 25 mL
ign. residue.................... <0.1% K................................... ≤0.005%
[26873-85-8] P4937-100ML 100 mL
chloride (Cl-)............... ≤0.05% Mg............................... ≤0.001%
P4937-500ML 500 mL
sulfate (SO42-)............. ≤0.05% Na................................... ≤0.01% A nonionic synthetic polymer of sucrose.
Al................................. ≤0.0005% NH4+.............................. ≤0.05% Used for cell separation and organelle isolation.
Ca................................... ≤0.001% Pb................................ ≤0.001%
 powder
Cu................................ ≤0.0005% Zn............................. ≤0.0005%
Fe................................. ≤0.0005% mol wt 300,000-550,000

F1418-25G 25 g
Similar to Ficoll® 400, but from a different supplier.
F1418-100G 100 g P7798-100G 100 g
P7798-500G 500 g
10

Ionic Metal Salts  Grade II, ≥98% Whole blood can be added directly to the
Solutions may contain slight haze. ACCUSPIN™ tube without the risk of mixing
Ionic gradient media, comprised of
concentrated heavy metal salts, are almost C6914-500G 500 g with the Histopaque®-1077 contained in the
C6914-1KG 1 kg lower chamber.
exclusively used for isopycnic separations of
nucleic acids.6 Cesium chloride and cesium
sulfate are the most widely used heavy Nonionic Iodinated Density Histopaque®-1077
 sterile-filtered, density: 1.077 g/mL
metal salts with gradient densities of up Gradient Media A solution containing polysucrose and sodium
to 1.91 g/cm3. Other useful salts include The iodinated aromatic compounds, diatrizoate, adjusted to a density of 1.077 g/mL.
sodium iodide, sodium bromide and the originally devised for X-ray contrast This medium facilitates the recovery of large
numbers of viable mononuclear cells.
rubidium salts. The steepness and shape of applications, solve the more serious
the ionic density gradient formed depends endotoxin.............................................................................................. tested
deficiencies of the other classes of gradient
on the centrifugal force and type of rotor media. Iodinated gradient media have
respectively. Ionic gradient media are highly much lower osmolarities and viscosities
ionic and non-viscous with high osmolarities. than sucrose at any concentration.
It should be kept in mind that the density Polysaccharides such as Ficoll® are even
of the sample is highly dependent on the more viscous than sucrose at all densities.
sample's hydration, which in turn depends Ionic gradient media, such as cesium
on the dehydration power of the ionic chloride, have higher densities and lower
gradient media. viscosities than other density gradient media.
However, their use is restricted due to the
Cesium chloride
high osmolarities and ionic strength which
[7647-17-8] CsCl FW 168.36
Used for the preparation of electrically conducting affect the hydration of osmotically sensitive
glasses.1,2 Used to make solutions for the particles and can disrupt or otherwise modify Histopaque® 10771-100ML
separation of RNA from DNA by density gradient the integrity of biological particles. store at: 2–8 °C
centrifugation.3
Because of their positive properties, 10771-100ML 100 mL
Lit cited: 1. Tver’yanovich, Y.S. et al., Glass Phys. Chem. 24, 446
10771-6X100ML 6 × 100 mL
(1998); 2. J. Am. Ceram. Soc. 90, 1822 (2007); 3. Molecular Cloning: iodinated gradient media are used in a wide
A Laboratory Manual , Cold Spring Harbor Laboratory Press (Cold 10771-500ML 500 mL
Spring Harbor, : 1989), range of applications. The structures of most
7.19-7.22; Histopaque®-1077 Hybri-Max™
iodinated compounds used as gradient
 Grade I, ≥99.0% media are based on tri-iodobenzoic acid  liquid, sterile-filtered, BioReagent, suitable for
Solutions may contain a slight haze. hybridoma
with hydrophilic groups attached to increase
C3011-25G 25 g
Used to create a density medium for the
their solubility. The first of these nonionic purification of lymphocytes and other
C3011-50G 50 g
density gradients, iohexol (e.g., Nycodenz® mononuclear cells.
C3011-100G 100 g
C3011-250G 250 g
and Histodenz™), became available in the endotoxin.............................................................................................. tested
C3011-500G 500 g 1970s. Iohexol solutions are more dense density............................................................................1.077 g/mL, 25 °C
store at: 2–8 °C
C3011-1KG 1 kg at any given concentration than the other
gradient media types. This means that a H8889-100ML 100 mL
 BioXtra, ≥99.5% (titration) H8889-500ML 500 mL
Insoluble matter.............................. passes filter test lower media concentration is needed for any
pH...5.0-7.5, 3 M H2O (20 °C) Fe................................. ≤0.0005% particular concentration which minimizes the Histopaque®-1083
sulfate (SO42-).......... ≤0.002% K...................................... ≤0.005% possibility of biological particles becoming  sterile-filtered, density: 1.083 g/mL
Al................................. ≤0.0005% Li................................... ≤0.0005% A solution containing polysucrose and sodium
dehydrated. Iohexol is nontoxic and not
As.............................. ≤0.00001% Mg.............................. ≤0.0005% diatrizoate, adjusted to a density of 1.083 g/mL.
Ba................................ ≤0.0005% Mn.............................. ≤0.0005% metabolized by mammalian cells.7
Facilitates the recovery of viable mononuclear cells
Bi.................................. ≤0.0005% Mo.............................. ≤0.0005% from rats, mice, and other small mammals.
Histopaque®
Ca................................... ≤0.005% Na..................................... ≤0.02%
endotoxin ............................................................................................. tested
Cd................................ ≤0.0005% Ni................................. ≤0.0005% Sigma Life Science offers a complete line of store at: 2–8 °C
Co................................ ≤0.0005% Pb................................ ≤0.0005% products for the separation or extraction of
Cr................................. ≤0.0005% Sr.................................. ≤0.0005% 10831-100ML 100 mL
leukocytes, viruses, DNA, RNA, organelles as 10831-6X100ML 6 × 100 mL
Cu................................ ≤0.0005% Zn................................. ≤0.0005%
well as many other applications. Featured
C3309-10G 10 g
in the product line is ACCUSPIN™, a sterile,
C3309-50G 50 g
C3309-250G 250 g 2-chamber tube separated by a porous frit.
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 11

Histopaque®-1119
 sterile-filtered, density: 1.119 g/mL
A solution containing polysucrose and sodium
diatrizoate, adjusted to a density of 1.119 g/mL.
Blood Centrifuge Plasma
Combined with Histopaque®-1077, it permits the
separation of mononuclear cells and granulocytes.
400 x g
endotoxin.............................................................................................. tested
store at: 2–8 °C
Mononuclear Cells
30 Minutes
11191-100ML 100 mL
Histopaque 1077
11191-6X100ML 6 × 100 mL Histopaque 1077
Red Cells
ACCUSPIN™ System-Histopaque®-1077
store at: 2–8 °C
Density Gradient Centrifugation using Histopaque®-1077
Gamma irradiated 12 mL polypropylene tubes An aliquot of Histopaque®-1077 medium is carefully layered with blood. The tube is then centrifuged (400 × g) for
fitted with a HDPE porous barrier and sterile-filled 30 minutes at room temperature. A visible layer of mononuclear cells forms at the Histopaque®-1077/plasma interface.
with 3 mL of Histopaque® 1077. Each tube will The solution above the mononuclear layer is carefully aspirated and discarded. The mononuclear layer is recovered,
separate 3-6 mL of anticoagulated blood. washed, and is ready for studies.
A6929-40X3ML 40 × 3 mL
Gamma irradiated 50 mL polypropylene tubes
fitted with a HDPE porous barrier and sterile-filled
with 15 mL of Histopaque® 1077. Each tube will
separate 15–30 mL of anticoagulated blood.
Plasma
Blood Centrifuge

Mononuclear cells
700 x g
Histopaque 1077

Histopaque 1077 30 Minutes Granulocytes

Histopaque 1119
Histopaque 1119

Red Cells

Density Gradient Centrifugation using Histopaque®-1119

This medium is used in conjunction with Histopaque®-1077 according to a double gradient technique. In this way a
layer of cells of the granulocytic series is separated from a zone containing lymphocytes, other mononuclear cells, and
platelets. Histopaque®-1077 is layered on top of Histopaque®-1119 followed by a layer of blood. After centrifuging (700
× g) for 30 minutes at room temperature, two distinct layers of cells become visibly evident. These separate fractions
are recovered by aspiration, washed, and are ready for granulocyte and mononuclear downstream applications.
A7054-12X15ML 12 × 15 mL
Gamma irradiated 50 mL polypropylene tubes
fitted with a HDPE porous barrier and sterile-filled
with 15 mL of Histopaque® 1077. Each tube will
separate 15–30 mL of anticoagulated blood.
Blood Centrifuge Plasma
A0561-100X15ML 100 × 15 mL

Mononuclear Cells
ACCUSPIN™ Tubes Sterile, 50 mL Capacity 1000 x g

Polypropylene radiation sterilized tube fitted with

a high density polyethylene barrier. Each tube will 10 Minutes Frit

accept 15 mL of density gradient. Frit

Histopaque
A2055-10EA 10 ea 1077
Red Cells
ACCUSPIN™ Tubes Sterile, 12 mL Capacity
Polypropylene radiation sterilized tube fitted with ACCUSPIN™ System-Histopaque®-1077 employs centrifuge tubes specially designed with two chambers separated
a high density polyethylene barrier. Each tube will by a porous high-density polyethylene barrier ("frit"). The lower chamber contains Histopaque®-1077 which allows the
addition of anticoagulated whole blood without risk of mixing with the separation medium. On centrifugation, the
accept 3 mL of density gradient.
whole blood descends through the frit to contact with the Histopaque®-1077 below the frit, giving a clear separation of
A1805-20EA 20 ea the blood components. The erythrocytes aggregate and the granulocytes become slightly hypertonic, increasing their
sedimentation rate, resulting in pelleting at the bottom of the ACCUSPIN™ tube. Lymphocytes and other mononuclear
cells, i.e., monocytes, remain at the plasma-Histopaque®-1077 interface.
12

Iodinated Compounds Meglumine diatrizoate Sodium diatrizoate hydrate

N-Methyl-D-glucamine diatrizoate
▼ Diatrizoic acid [131-49-7] C7H17NO5 · C11H9I3N2O4 FW 809.13
O ONa
• xH2O
I I
O O
O OH M5266-10G 10 g
M5266-25G 25 g H 3C N N CH3
I I H H
O O I
M5266-100G 100 g
H3C N N CH3 3,5-Diacetamido-2,4,6-triiodobenzoic acid sodium
H H
I salt; Diatrizoic acid sodium salt hydrate; 3,5-Bis(acetyl-
OptiPrep™ Density Gradient Medium amino)-2,4,6-triiodobenzoic acid, sodium salt
Diatrizoic acid O CH3 O CH3 C11H8I3N2NaO4 · xH2O FW 635.90 (Anh)
O I OH I O
Amidotrizoic Acid  ≥99%
N N
[117-96-4] C11H9I3N2O4 FW 613.91 HO N
H
N
H
OH
OH OH S4506-10G 10 g
D9268-50G 50 g I I I I
S4506-50G 50 g
HO N O O N OH
H H S4506-100G 100 g
Diatrizoic acid dihydrate OH OH
S4506-250G 250 g
[50978-11-5] C11H9I3N2O4 · 2H2O FW 649.94 Iodixanol solution S4506-500G 500 g
 meets USP testing specifications 60% (w/v) solution of iodixanol in water (sterile).
References:
D9809-10G 10 g Density gradient suitable for the isolation of cells 1. Biological Centrifugation, J. Graham, p. 5
and cell organelles. 2. Laboratory Techniques in Biochemistry and Molecular Biol-
Diatrizoic acid ▲ ogy, P.T. Sharpe, p.24
D1556-250ML 250 mL
3. ibid., p.26
Histodenz™
4. Biological Centrifugation, J. Graham, p. 24
5-(N-2,3-Dihydroxypropylacetamido)-2,4,6-triiodo- 5. Centrifugation, A Practical Approach (2nd Edition),
N,N'-bis(2,3-dihydroxypropyl)isophthalamide D. Rickwood, p.37
6. ibid., p.38
[66108-95-0] C19H26I3N3O9 FW 821.14
7. Laboratory Techniques in Biochemistry and Molecular Biol-
O I O ogy, P.T. Sharpe, p.33
HO N N OH
H H
OH OH
I I
N CH3

HO O
OH

Useful as a nonionic density gradient medium.

store at: room temp

D2158-100G 100 g
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 13

Selection Table
Name Applications Cells Organelles Viruses Macromolecules
ACCUSPIN™ Suitable for separation of mononuclear cells from human •
peripheral blood or bone marrow.
Cesium chloride Used to make solutions for the separation of RNA from DNA by • •
density gradient centrifugation. May be used for the separation
of ribosomal subunits, proteins, glycoproteins, and viruses.
Dextran Used to prepare leukocyte rich plasma. •
Dextran sulfate Used to precipitate lipoproteins. •
Diatrizoic acid Used with polysucrose 400 to produce density gradients •
capable of purifying living cells and cell fragments.
Ficoll® A nonionic synthetic polymer of sucrose. Used for cell • •
separation and organelle isolation. See also Polysucrose 400.
Histodenz® Useful as a nonionic density gradient medium. Autoclavable, • • • •
universal centrifugation medium recommended for isolation of
macromolecules, nucleoproteins, cell organelles, and a variety
of cell types and viruses.
Histopaque® 1077 Suitable for separation of mononuclear cells from human •
peripheral blood or bone marrow. In cell culture, used to
separate live from dead cells.
Histopaque® 1083 Suitable for separation of mononuclear cells from rat, mouse, •
or other mammalian peripheral blood or bone marrow.
Histopaque® 1119 Used in conjunction with Histopaque 1077 for the separation •
of granulocyes and mononuclear cells from human peripheral
blood or bone marrow.
Meglumine Used in conjunction with sodium diatrizoate to separate a •
diatrizoate variety of cell types and bacterial spores.
OptiPrep™ Density 60% (w/v) solution of iodixanol in water (sterile). • • • •
Gradient Medium Density gradient suitable for the isolation of cells and cell
organelles, as well as viruses and proteins.
Percoll® Possesses nearly ideal physical characteristics for the separation • • • •
of cells, viruses, organelles and other subcellular particles.
Percoll is isosmotic throughout the gradient, non-toxic and is
easily removed from the purified materials. It is supplied as a
sterile colloidal suspension comprised of silica particles,
15-30 nm in diameter, coated with polyvinylpyrrolidone (PVP).
Polysucrose 400 Nonionic synthetic polymer of sucrose. Used for cell • •
separation and organelle isolation. May be used to increase
the sedimentation rate of red blood cells. Acts as a stabilizing
agent in protein solutions. Useful in preparing density
gradients and high density solutions.
Sodium diatrizoate Used with polysucrose 400 to produce density gradients •
capable of purifying living cells and cell fragments. May also
be used to obtain complete leukocyte suspensions and to
isolate and prepare pure thrombocytes and reticulocytes.
Sucrose Used to prepare gradients for various rate-zonal and isopycnic •
procedures. May be used in the purification of proteins,
nucleic acids, ribosomes and polysomes. May be used for cell
separation when viability is not essential.
14

Histopaque® Troubleshooting Guide

Histopaque® Troubleshooting Guide

The following recommendations for when using Histopaque but is not meant to • Difficulty in separating mononuclear
troubleshooting the use of Histopaque and be a comprehensive list. or polymorphonuclear cells from rat or
ACCUSPIN™ in cell separation techniques mouse blood
Typical problems observed when
have been compiled by Sigma-Aldrich® • Low recovery
Technical Services. These are based on using Histopaque/ACCUSPIN • Red blood cell, platelet, or neutrophil
observations and experience from resolving • Difficulty in separating mononuclear or contamination in separated cells
customer problems. This guide addresses polymorphonuclear cells from human blood
the most common sources of error observed

Blood or Cell Samples Troubleshooting Tips

Cause Solution
Blood drawn >24 hours prior to Use blood drawn from 2–6 hours prior to separation. Blood drawn >24 hours will be more difficult to separate, and percent
separation recovery and viability will be lower.
Blood has coagulated due to Use of vacuum tubes with premeasured amounts of anticoagulant is suggested. Use of a syringe (without anticoagulant) is not
absence of anticoagulant recommended.
Blood has coagulated due to Ensure vacuum tubes with anticoagulant are mixed well after blood draw.
incomplete mixing
Anticoagulant used is not suitable The following anticoagulants are suitable for use with Histopaque:
for use in Histopaque separation
application • 15–30 units heparin per mL whole blood
• 1.25–1.75 mg EDTA per mL whole blood
ACA-A (Acid Citrate Dextrose formula A) may be used as an anticoagulant. After separation, the mononuclear cell band produced
will be wider than for other anticoagulants. This is normal and the cells are acceptable for use.
Blood sample is too warm and Red blood cell contamination may be a result of blood not being at room temperature.
separation attempted too soon after
blood draw After drawing, the blood should be allowed to cool at room temperature for minimum 30-45 minutes. If used immediately after
being drawn, the number of mononuclear cells collected will be low. Separation procedures are optimal when both blood and
Histopaque are at 18–20 °C, with an acceptable temperature range of 18–26 °C.
Blood sample diluted with high salt The historic cell separation technique used with Histopaque includes dilution of the blood with PBS (1:1). Subsequently, we have
concentration PBS found not all PBS formulations are suitable for use with Histopaque.

Solutions containing 150 mM phosphate buffer and 150 mM sodium chloride are PBS solutions with salt concentrations too high
for use with Histopaque. PBS molarities closer to 10 mM phosphate are more suitable for use with Histopaque. Cell viability will
remain higher when high salt concentration PBS is replaced by a suitable cell culture media.
Differences in donor blood High lipid levels, rheumatoid factor, anemia, and drug treatment are all possible causes for poor separation of a specific
physiology (when single sample has donor’s blood.
poorer recovery)
If the plasma is not clear, this is an indication of high lipid levels.
Contamination of small animal Blood obtained from a non-needle bleed, e.g., through tail sampling, is often unacceptable for use due to the presence of hairs
sample with other fluids or solids and other body fluids in the collected blood. This often results in the coagulation cascade being initiated, producing clotting.

Recommend the animal be sacrificed and the blood obtained through either the aorta or vena cava or other large blood vessel.
Alternatively, a pediatric vacuum tube may be used for drawing blood.
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 15

Blood or Cell Samples Troubleshooting Tips—Cont'd

Cause Solution
Occlusion of white blood cells in Aggregates may form when red cells come in contact with the polysucrose, resulting in occlusion of white blood cells.
samples with high levels of white Once trapped in these aggregates, the white blood cells will travel to the bottom of the centrifuge tube.
cells
For routine blood specimens, percent recovery will often be higher for undiluted blood. However, bone marrow, umbilical cord
blood, and samples from donors with abnormally high white cell counts should be diluted to reduce the size of the red cell
aggregates and improve recovery of white cells.
Improper dilution of blood or When blood is diluted prior to loading on the Histopaque®, there is a chance either the buffer or media formulation is
dilution with contaminated PBS or incompatible with Histopaque, or that the PBS or cell culture media is contaminated.
media
If the blood has been diluted and yields are poor, repeat experiment without sample dilution.
Dilution using cell culture media Cell culture media without FBS can be substituted for PBS when diluting samples for use with Histopaque. If the blood is diluted
containing FBS or other carrier with cell culture media containing FBS, the number of cells recovered will be lower.
protein
FBS may be added to the culture media for subsequent washing or storage after the cells have been separated and washed at
least once to remove the Histopaque. After the first wash, adding protein such as FBS to the wash media will further protect the
cells from damage.
Isolating cells from buffy coats using A buffy coat is prepared by collecting blood in a suitable anticoagulant and centrifuging. Red cells will pellet to the bottom of the
Histopaque tube. Directly on top of the red cell layer will be a gray layer. This gray layer is the buffy coat and represents all the white blood
cells in the blood. Above the gray layer will be the plasma.

Buffy coats prepared from fresh blood are suitable for separation using Histopaque. If the cells are several days old, e.g., if the buffy
coats are obtained from donated units of blood, cells may not provide acceptable yields.

Buffy coats must be diluted a minimum of 1:2 or 1:4 before being loaded onto the Histopaque gradient.
Isolating cells from buffy coats using Buffy coat preparations cannot be used in ACCUSPIN tubes. The ACCUSPIN tubes require a certain quantity of red blood cells to
ACCUSPIN™ tubes work properly, and buffy coats lack sufficient red blood cells for use in ACCUSPIN tubes

Histopaque/ACCUSPIN Troubleshooting Tips

Cause Solution
Bacterial contamination of Histopaque is manufactured as a sterile solution. There are no preservatives present to reduce chance of contamination. If
Histopaque contamination is suspected, use fresh bottle of Histopaque.
Histopaque temperature is too cold If Histopaque or filled ACCUSPIN tubes are used cold, yields are typically very low and clumping of the cells may be observed. Red
blood cell contamination may also be a result of Histopaque not being at room temperature.

Temperature is extremely important when performing the procedure. A 100 mL or 500 mL bottle of Histopaque stored at 2–8 °C
may take several hours to reach 18–20 °C. When planning to use Histopaque, we recommend removing the Histopaque from the
refrigerator the previous day and let the bottles stand on the bench overnight. This ensures the solution is at room temperature
and ready for use.

Another option is to transfer the proper amount of Histopaque to each centrifuge or ACCUSPIN tube and allow the smaller
volumes to acclimate to room temperatures.

After the cell band has been collected and the first wash performed, it is acceptable to perform the remaining wash steps at 4 °C.
16

Technique Troubleshooting Tips

Cause Solution
Delay in processing samples after When layering the blood onto the gradients intended for mononuclear cells, precise layering is not required. If a little mixing
layering blood on Histopaque occurs between the blood and the Histopaque, the cell band will still form properly.

Once the blood has been layered, it is important to immediately place the centrifuge tubes into the centrifuge. If too much
time elapses, the entire layer of Histopaque will be tinged red from the red blood cells. Depending upon the amount of “droplet
formation,” this may lower the percent recovery of mononuclear cells. The number of tubes processed at one time should be
limited to reduce this cell dispersal.
Improper layering of Histopaque® When using both Histopaque 1077 and Histopaque 1119 to isolate neutrophils, careful technique must be used to prevent mixing
1077 and Histopaque 1119 when at the solution interface.
isolating neutrophils
Check the integrity of the interface using Schlieren optics. Any swirling or mixing at the interface between the layers should be
evident when holding the tube up against the light. If the interface was properly prepared, there should be a sharply demarcated
line.

If the sharply defined line is not present or if swirling is present at the interface, discard the tube and start over.

It is also important to use the gradient as soon as it is formed. There are no chemical differences between 1077 and 1119; the two
solutions have the same components and will start diffusing together over time. When this happens, recoveries will be poor or
nonexistent.
Contamination from powder used in Powdered gloves should not be used. The glove powder causes the cells to aggregate. Use powder free gloves only.
powdered gloves
Incorrect centrifugation force used The centrifuge should be checked to ensure proper calibration. Excessive force (higher centrifugation speed) will lower yield.
Histopaque was autoclaved to Histopaque cannot be sterilized by autoclaving; autoclaving will caramelize the polysucrose in the solution, turning it brown.
sterilize prior to use Sterile filtering Histopaque through a 0.22 micron filter is acceptable.
Platelet or neutrophil contamination Platelet contamination or neutrophil contamination is generally the result of collecting too much plasma or Histopaque 1077 or
in cells after separation 1083 when the cell band is being collected. Reduce the amount of plasma or Histopaque collected.
Too large a volume used to wash Washing the cell pellet typically removes platelets. If a large amount of wash solution is left over the cell pellet, this results in more
cell pellet platelets in the cell suspension.

Remove as much wash media as possible without disturbing the cell pellet. Platelets normally do not pellet below a centrifugal
force of 1000 × g for 10 minutes. At the lower centrifugation speeds recommended for the wash steps, the platelets should stay
suspended in the wash media or supernatant.
Cell pellet does not form single cell When resuspending the cell pellet, only a small amount of wash solution should be used. We recommend no more than 0.5 mL
suspension wash media when performing the procedure in a 15 mL or 50 mL centrifuge tube. This wash media is gently rinsed over the pellet.

It may be necessary to draw the cell suspension into the pipet and dispense several times. Once the cells are in a single cell
suspension, the remaining wash solution can be added and the solution centrifuged.
Loss of cells during washing due to Occasionally cells stick to the walls of the centrifuge tube. If the cells do adhere to the polystyrene tube, the tube will appear to
adhesion to centrifuge tube wall become cloudy.

Change to another lot of polystyrene centrifuge tubes or switch to another type of plastic such as polyethylene. Polystyrene tubes
are most commonly observed to cause this problem, but other types of plastic have also been reported to have cell adhesion.
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 17

Cell Viability and Proliferation

Cell Viability and Proliferation

Assays to measure proliferation, viability, Cell Viability Kits In Vitro Toxicology Assay Kit, Lactic
and cytotoxicity are commonly used to Dehydrogenase based
monitor the response and health of cells in Cell Counting Kit LDH reduces NAD+, which then converts a
culture after treatment with various stimuli.  sufficient for 500 tests tetrazolium dye to a soluble, colored formazan
The Cell Counting Kit (CCK) is used for the derivative.
The proper choice of an assay method
fluorometric detection of living cell numbers. The For spectrophotometric measurement of viable
depends on the number and type of cells
amount of a fluorescent dye, calcein, produced cells. Absorbance of converted dye is measured at a
used as well as the expected outcome. from calcein-AM by esterases in cells is directly wavelength of 490 nm.
Assays for cell proliferation may monitor proportional to the number of viable cells in a 1 kit sufficient for 500 tests
the number of cells over time, the number culture media. store at: −20 °C
of cellular divisions, metabolic activity, or Components TOX7-1KT 1 kit
DNA synthesis. Cell counting using viability Calcein-AM reagent
DPBS In Vitro Toxicology Assay Kit, MTT based
dyes such as trypan blue or calcein-AM can Protocol
provide both the rate of proliferation as well store at: −20 °C Conversion of MTT to a water-insoluble colored
as the percentage of viable cells. formazan derivative which is then solubilized in
03285-1KT-F 1 kit
acidic isopropanol.
5(6)-Carboxyfluorescein diacetate Cell Counting Kit - 8 For spectrophotometric measurement of cell viability
N-succinimidyl ester (CFSE) is a popular by mitochodrial dehydrogenase. Absorbance of
Cell Counting Kit-8 (CCK-8) allows convenient converted dye is measured at a wavelength of
choice for measuring the number of cellular assays using WST-8 (2-(2-methoxy-4-nitrophenyl)- 570 nm.
divisions a population has undergone. 3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-
tetrazolium, monosodium salt), which produces 1 kit sufficient for 1,000 tests
Upon entering the cell, CFSE is cleaved by
a water-soluble formazan dye upon bioreduction store at: 2–8 °C
intracellular esterases to form the fluorescent
in the presence of an electron carrier, 1-Methoxy TOX1-1KT 1 kit
compound and the succinimidyl ester group PMS. CCK-8 solution is added directly to the
covalently reacts with primary amines on cells, no pre-mixing of components is required. In Vitro Toxicology Assay Kit, Neutral Red
intracellular proteins. Upon division, the WST-8 is bioreduced by cellular dehydrogenases based
to an orange formazan product that is soluble in
fluorescence intensity of each daughter Neutral red is taken up by viable cells and stored in
tissue culture medium. The amount of formazan
cell is halved which allows for the simple the lysosomes. The dye is extracted and the uptake is
produced is directly proportional to the number of
detection of the number of cell divisions by quantitated by spectroscopy.
living cells. Since the CCK-8 solution is very stable
flow cytometry. and it has little cytotoxicity, a longer incubation, For spectrophotometric measurement of viable
such as 24 to 48 hours, is possible. cells. Absorbance of converted dye is measured at a
Assays that measure metabolic activity are Cell Counting Kit-8 allows sensitive colorimetric
wavelength of 540 nm.
suitable for analyzing proliferation, viability, assays for the determination of the number of 1 kit sufficient for 1,000 tests
and cytotoxicity. The reduction of tetrazolium viable cells in the proliferation and cytotoxicity store at: 2–8 °C

salts such as MTT and XTT to colored assays. The detection sensitivity is higher than any TOX4-1KT 1 kit
other tetrazolium salts such as MTT, XTT or MTS.
formazan compounds or the bioreduction of store at: −20 °C In Vitro Toxicology Assay Kit, Resazurin
resazurin only occurs in metabolically active
96992-500TESTS-F 500 test based
cells. Actively proliferating cells increase their 96992-3000TESTS-F 3000 test Bioreduction of the dye reduces the amount of its
metabolic activity while cells exposed to oxidized form (blue) and concomitantly increases the
toxins will have decreased activity. fluorescent intermediate (red).
For spectrophotometric measurement of metabolic
activity of living cells. Absorbance of converted dye
is measured at a wavelength of 600 nm.
1 kit sufficient for 2,000 tests
store at: 2–8 °C

TOX8-1KT 1 kit
18

In Vitro Toxicology Assay Kit, spectrophotometrically yielding absorbance as

Sulforhodamine B based a function of concentration of converted dye.
Dye binds to cellular protein and is then
The cleavage and conversion of the soluble
solubilized in base.
yellow dye to the insoluble purple formazan
For spectrophotometric measurement of biomass
(viable and non-viable cells) by total protein. has been used to develop an assay system
Absorbance of dye is measured at a wavelength alternative to the conventional 3H-thymidine
of 565 nm. uptake and other assays for measurement
1 kit sufficient for 1,000 tests of cell proliferation. Active mitochondrial
TOX6-1KT 1 kit dehydrogenases of living cells will cause
this conversion. Dead cells do not cause this
In Vitro Toxicology Assay Kit, XTT based A. Human Foreskin Fibroblasts passage 28 (senescent cells)
change. This has been applied in measurement
Conversion of XTT to a water-soluble colored of interleukin-2 activity in a multiwell assay.2
formazan derivative.
Modification has improved the sensitivity.3
For spectrophotometric measurement of
cell viability by mitochodrial dehydrogenase.
Other uses such as measurement of
Absorbance of converted dye is measured at a cytotoxicity4 and cell number have also been
wavelength of 450 nm. developed.
1 kit sufficient for 1,000 tests In our testing we dissolve MTT, (Cat. No.
store at: −20 °C
M5655), 5 mg/ml in RPMI-1640 without
TOX2-1KT 1 kit
phenol red. This medium is available as a
Live/Dead Cell Double Staining Kit powder (Cat. No. R8755) or liquid (Cat. No.
Staining kit for live/dead cells The Live/Dead
R7509). The solution is filtered through a
B. Human Foreskin Fibroblasts passage 5 (control)
Cell Double Staining Kit contains calcein-AM 0.2 μm filter and stored at 2–8 °C for frequent
and propidium iodide (PI) solutions for the Detection of senescent cells. Primary Human Foreskin use or frozen for extended periods.
simultaneous fluorescent detection of viable and Fibroblasts (HFF) at early and late passages (5 and 28
dead cells in cell cultures. passages, respectively) were stained using the Senescent Routinely, MTT stock solution (5 mg/ml)
Cell Staining Kit (Prod. No. CS0030). The HFF cells at is added to each culture being assayed to
passes test for fluorescence passage 28 show a blue staining indicating that these cells
Components are senescent, (A) whereas at passage 5, senescent cells equal one-tenth the original culture volume
Solution A (Calcein AM solution) 4 × 50 μl are absent. (B) and incubated for 3 to 4 hr. At the end of
Solution B (propidium iodide solution) 300 μl Components the incubation period the medium can be
store at: −20 °C
X-gal solution 4 mL removed if working with attached cells and
04511-1KT-F 1 kit Staining Solution, 10 x 15 mL the converted dye may be solubilized with
Fixation Buffer 10 x 15 mL
Senescence Cells Histochemical Staining Reagent B 1.5 mL acidic isopropanol (0.04-0.1 N HCl in absolute
Kit Reagent C 1.5 mL isopropanol). When working with suspension
Dulbecco's Phosphate Buffered Saline (PBS) 10x 60 mL
 sufficient for 100 tests store at: −20 °C
cells the dye is added directly and dissolution
Replicative senescence is a growth-arrest is accomplished by trituration. Absorbance of
CS0030-1KT 1 kit
state associated with loss of division potential, converted dye is measured at a wavelength
changes in cell morphology, shape and physical of 570 nm with background subtraction at
appearance, and the pattern of gene expression MTT 630–690 nm.
in cells.
Cell Viability Applications
Histochemical staining of β-galactosidase activity MTT may also be used to score hybridoma
is performed at pH 6.0. Under these conditions, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- development or clonal development. Clones
β-galactosidase is a biomarker specific for diphenyltetrazolium bromide; thiazolyl blue) will convert the dye and become readily visible
senescent cells, but is not found in quiescent, is a water soluble tetrazolium salt yielding a
immortal, or tumor cells. without magnification. It should be noted
yellowish solution when prepared in media that MTT is a mutagen and the resultant cells
Manufactured under license to US Patent Nos.
5,491,069 and 5,795,728.
or salt solutions lacking phenol red. Dissolved may be affected. The concentration used may
MTT is converted to an insoluble purple be reduced by dilution of the stock solution
formazan by cleavage of the tetrazolium (5 mg/ml) to 0.1 mg/ml and adding a tenth
ring by dehydrogenase enzymes.1 This volume to each well. Incubation should be
water insoluble formazan can be solubilized monitored by observing for stained clones.
using isopropanol or other solvents Cells can be recovered by gently washing the
and the dissolved material is measured cells and adding growth medium.
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 19

Technique Summary Trypan Blue 5. Repeat this procedure for chamber 2.

MTT stock solution: 5 mg/mL Trypan Blue is one of several stains Note: If greater than 10% of the cells appear
Typical use: Add 1/10th of culture volume recommended for use in dye exclusion clustered, repeat entire procedure making
Solvent: 0.04-0.1 N HCl in isopropanol procedures for viable cell counting. This sure the cells are dispersed by vigorous
Can also use DMSO:isopropanol (1:1) method is based on the principle that pipetting in the original cell suspension
Spectrophometric reading: 570 nm live (viable) cells do not take up certain as well as the Trypan Blue-cell suspension
Background wavelength: 630-690 nm dyes, whereas dead (non-viable) cells do. mixture. If less than 200 or greater than
Staining facilitates the visualization of cell 500 cells (i.e., 20–50 cells/square) are
References
1. Slater, T.F., et al., Biochim. Biophys. Acta, 77, 383 (1963).
morphology. observed in the 10 squares, repeat the
2. Mossman, T., J. Immunol. Methods, 65, 55 (1983).
NOTE: Trypan Blue has a greater affinity for procedure adjusting to an appropriate
3. Denizot, F. and Lang, R., J. Immunol. Methods, 89, 271 dilution factor.
(1986). serum proteins than for cellular protein. If
4. Carmichael, J., et al., Cancer Research, 47, 936 (1987). the background is too dark, cells should be 6. Withdraw a second sample and repeat
Thiazolyl Blue Tetrazolium Bromide pelleted and resuspended in protein-free count procedure to ensure accuracy.
MTT; 3-(4,5-Dimethyl-2-thia- CH3 medium or salt solution prior to counting.
N Calculations
zolyl)-2,5-diphenyl-2H-tetra-
zolium bromide; Methylthia-
Br
N N S CH3 Protocol for Viable Cell Counting using Cell Counts - Each square of the
zolyldiphenyl-tetrazolium N
N Trypan Blue hemacytometer, with cover-slip in place,
bromide [298-93-1] C18H16BrN5S
1. Prepare a cell suspension in a balanced salt represents a total volume of 0.1 mm3
FW 414.32
solution (e.g., Hanks' Balanced Salts [HBSS], or 10-4 cm3. Since 1 cm3 is equivalent to
 98%
store at: 2–8 °C Cat. No. H9269). approximately 1 ml, the subsequent cell
M2128-100MG 100 mg 2. Transfer 0.5 ml of 0.4% Trypan Blue solution concentration per ml (and the total number
M2128-250MG 250 mg (w/v) to a test tube. Add 0.3 ml of HBSS of cells) will be determined using the
M2128-500MG 500 mg
and 0.2 ml of the cell suspension (dilution following calculations:
M2128-1G 1 g
M2128-5G 5 g factor = 5) and mix thoroughly. Allow to Cells Per mL = the average count per square
M2128-10G 10 g stand for 5 to 15 minutes. × dilution factor × 104 (count 10 squares)
 powder, BioReagent, suitable for cell culture, Note: If cells are exposed to Trypan Blue for Example: If the average count per square is
suitable for insect cell culture, ≥97.5% (HPLC) extended periods of time, viable cells, as well 45 cells × 5 × 104 = 2.25 × 106 cells/ml.
store at: 2–8 °C
as non-viable cells, may begin to take up dye.
M5655-5X1G   Total Cells = cells per ml × the original
M5655-100MG 100 mg 3. With the cover-slip in place, use a Pasteur
volume of fluid from which cell sample was
M5655-500MG 500 mg pipette or other suitable device to transfer
M5655-1G 1 g removed.
a small amount of Trypan Blue-cell
suspension mixture to both chambers of Example: 2.25 × 106 (cells/ml) × 10 ml
RPMI-1640 Medium
the hemacytometer. Carefully touch the (original volume) = 2.25 × 107 total cells.
 Modified, with sodium bicarbonate, without
edge of the cover-slip with the pipette tip
L-glutamine and phenol red, liquid, sterile-filtered, Cell Viability (%) = total viable cells
suitable for cell culture and allow each chamber to fill by capillary
(unstained) ÷ total cells (stained and
endotoxin.............................................................................................. tested action. Do not overfill or underfill the
store at: 2–8 °C unstained) × 100.
chambers.
R7509-500ML 500 mL Example: If the average count per square
R7509-6X500ML 6 × 500 mL 4. Starting with chamber 1 of the
of unstained (viable) cells is 37.5, the total
R7509-24X500ML 24 × 500 mL hemacytometer, count all the cells in the
viable cells = [37.5 × 5 × 104] viable cells/
R7509-1L 1 L 1 mm center square and four 1 mm corner
R7509-6X1L 6 × 1 L ml × 10 ml (original volume) = 1.875 × 107
squares (see Diagram I). Non-viable cells
viable cells. Cell viability (%) = 1.875 × 107
 Modified, with L-glutamine, without phenol red will stain blue. Keep a separate count of
and sodium bicarbonate, powder, suitable for cell (viable cells) ÷ 2.25 × 107 (total cells) × 100 =
viable and non-viable cells.
culture 83% viability.
Formulated to contain 10.4 grams of powder per Note: Count cells on top and left touching
liter of medium. middle line of the perimeter of each square.
Supplement with 2.0 g/L sodium bicarbonate. Do not count cells touching the middle line
store at: 2–8 °C
at bottom and right sides (see Diagram II).
R8755-10X1L 10 × 1 L
R8755-10L 10 L
20

Counting Cells Using Hemocytometer Trypan Blue solution Neutral Red Solution (0.33%)
 0.4%, liquid, sterile-filtered, suitable for cell culture
DIAGRAM I Neutral Red solution; H3C N Cl
STANDARD HEMOCYTOMETER CHAMBER Prepared in 0.81% sodium chloride and 0.06% Toluylene red; 3-Amino-7- CH3
potassium phosphate, dibasic. dimethylamino-2-methyl-
H2N N
H
N
CH3
T8154-20ML 20 mL phenazine hydrochloride
T8154-100ML 100 mL [553-24-2] C15H17ClN4
FW 288.78
Trypan Blue ▲  3.3 g/L in DPBS, sterile-filtered, BioReagent,
suitable for cell culture

Stains, Dyes, and Indicators Can be used as a vital stain, to stain living cells.
endotoxin.............................. tested
Giemsa stain store at: 2–8 °C

Azure mixture sicc. Giemsa stain N2889-20ML 20 mL

[51811-82-6] C14H14ClN3S FW 291.80 N2889-100ML 100 mL
 powder, BioReagent, suitable for cell culture
Used as a chromosome stain and to differentiate Nigrosin
nuclear morphology of platelets, RBCs, WBCs, and Acid black 2; Nigrosin water soluble [8005-03-6]
The circle indicates the approximate area other cell types.  certified by the Biological Stain Commission
covered at 100× microscope magnification G9641-5G 5 g Certified for use in Dorner's spore stain. Used as a
(10× ocular and 10× objective). Include cells G9641-25G 25 g negative stain in place of India ink for spirochetes,
bacteria, protozoa and fungi.
on top and left touching middle line (O).  certified by the Biological Stain Commission
198285-25G 25 g
Do not count cells touching middle line at Certified for Giemsa stain for blood films and for
198285-100G 100 g
bottom and right (Φ). Count 4 corner squares the Wolbach's Giemsa method for paraffin sections.
and middle square in both chambers (one G5637-5G 5 g Nigrosin water soluble
G5637-25G 25 g
chamber represented here). Acid black 2 [8005-03-6]

DIAGRAM II Neutral Red  powder, BioReagent, suitable for cell culture

CORNER SQUARE (ENLARGEMENT)
Used as a viable cell staining agent especially in
3-Amino-7-dimethyl- H3C N Cl
stem cell biology.
amino-2-methyl- CH3
H2N N N
phenazine hydrochloride; H N4763-25G 25 g
CH3
Toluylene red; Basic Red
5 [553-24-2] C15H17ClN4 ▼ Phenol Red
FW 288.78
Suitable for use as a pH indicator.
Useful as an indicator for preparing neutral red
paper, and as a biological stain. Phenol Red
Phenolsulfonphthalein [143-74-8] HO
▼ Trypan Blue  powder, BioReagent, suitable for cell culture
C19H14O5S FW 354.38 OH
composition
H3C CH3
NH2 OH OH NH2   Dye content ≥90%
N N N N O
S
O O O O Purified O O
NaO S S ONa NaO S S ONa
store at: 2–8 °C
O O O O  powder, BioReagent, suitable for cell culture
[72-57-1] C34H24N6Na4O14S4 FW 960.81 N4638-1G 1 g P3532-5G 5 g
N4638-5G 5 g P3532-25G 25 g
Use to detect dead and dying cells in cytotoxicity
assays and for routine assessment of cell viability.  certified by the Biological Stain Commission
Certified for use in the method of Doan & Ralph for
Phenol Red sodium salt
Trypan Blue the supravital staining of living blood cells. Phenolsulfonephthalein sodium
O
Direct blue 14 C34H24N6O14S4Na4 salt [34487-61-1] C19H13NaO5S
composition FW 376.36
S ONa
O
 powder, BioReagent, suitable for cell culture   Dye content 50-60%
composition 861251-25G 25 g HO O

  Dye content ~40%  powder, BioReagent, suitable for cell culture,

suitable for insect cell culture
T6146-5G 5 g
T6146-25G 25 g P5530-5G 5 g
T6146-100G 100 g P5530-25G 25 g
P5530-50G 50 g

Phenol Red ▲
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 21

Resazurin sodium salt  certified by the Biological Stain Commission Hemacytometer

7-Hydroxy-3H-phenoxazin-3-one- O O ONa composition
10-oxide sodium salt [62758-13-8]   Dye content 75% Bright-Line™ Hemacytometer
N
C12H6NNaO4 FW 251.17 H-shaped moat forms two cell-counting areas.
O 199303-1G 1 g
199303-5G 5 g The surface features enhanced Neubauer rulings.
For the measurement of metabolic activity and
199303-25G 25 g Replacement cover slips sold separately.
proliferation of living cells.1,2 The bioreduction of
the dye reduces the amount of the oxidized form Supplied with two cover slips.
(blue) and concomitantly increases the fluorescent Sulforhodamine B sodium salt
intermediate (red).
[3520-42-1] ONa
Lit cited: 1. Ahmed, S.A., et al., A new rapid and simple non- O S O
radioactive assay to monitor and determine the proliferation of
C27H29N2NaO7S2
lymphocytes: an alternative to [3H]thymidine incorporation assay. J. FW 580.65 O
Immunol. Methods 170, 211-24 (1994); 2. DeFries, R., and Mitsuhashi, S O
M., Quantification of mitogen induced human lymphocyte O
proliferation: comparison of alamar blue assay to 3H-thymidine
incorporation assay. J. Clin. Lab Anal. 9, 89-95 (1995); H3C N O N CH3

 powder, BioReagent, suitable for cell culture H3C CH3

composition  powder, BioReagent, suitable for cell culture Z359629-1EA 1 ea

  Dye content ~80% Use as a fluorescent dye to quantify cellular proteins
R7017-1G 1 g in cultured cells. Bright-Line™ Hemacytometer replacement
R7017-5G 5 g composition cover slip
  Dye content ~75% Z375357-1EA 1 ea
S1402-1G 1 g
S1402-5G 5 g
S1402-25G 25 g

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22

Organelle Isolation

Organelle Isolation
The isolation of subcellular fractions Lysosomes are organelles ubiquitous in most
• Compatible with products for structure prokaryotic and eukaryotic cells. They contain
by centrifugation is a commonly used confirmation—easily confirm intactness with
many acid hydrolases that take part in protein
technique and is widely applicable across companion kit test for cytochrome C reductase
degradation in the cell. Lysosomes also contain
multiple cell and tissue types. Because (CY0100)
lipases, nucleases, and polysaccharidases, and
organelles differ in their size, shape, and • Can be used to isolate ER from soft animal deficiencies in some of these enzymes lead
to specific lysosomal storage diseases such as
density, centrifugation can be easily tissues and cultured cells
Tay Sachs, Gaucher, Hunter disease and others.
employed to separate and purify organelle Components
They also contribute to maintaining cellular
fractions from gently homogenized samples. Isotonic Extraction Buffer 5X 100 mL homeostasis. Malfunctions in this organelle directly
Hypotonic Extraction Buffer 10 mL
To be suitable for further analysis, isolated Calcium chloride, 2.5 M solution 5 mL
impact cell behavior and fate. Lysosomes may also
organelles must be relatively pure, and OptiPrep™ Density Gradient Medium (Sigma D1556) be involved in other cellular processes such as
100 mL Albinism and aging.
structurally and functionally intact. It is also
Blunt Nosed Needle 1 ea
important to verify the organelle integrity store at: 2–8 °C • Generates functional organelles for metabolism
with robust assays. and disease research
ER0100-1KT 1 kit
Sigma Life Science offers several organelle Golgi Isolation Kit • Includes protease inhibitor to manage
isolation and enrichment kits, companion degradation
 sufficient for 50 g (tissue)
organelle functionality assays, and antibodies The Golgi Isolation Kit provides a method for • Measures integrity with included Neutral Red dye
that are suitable for evaluation of samples isolating Golgi membranes from mammalian soft
from plant, animal, and cell culture sources. tissues by discontinuous density gradient. • Choose from multiple options to obtain enriched
fractions
The Golgi Isolation kit was optimized using rat liver
Density Gradient Kits and tested on rat kidney, spleen, and heart. • Enriched fractions for intact and functional
Components lysosomes from tissues and cells
Endoplasmic Reticulum Isolation Kit Isolation Buffer 5X 120 mL
2.3 M Sucrose Solution 120 mL
• Suitable for functional studies including protein
 isolation of intact ER from mammalian soft tissues degradation
Protease Inhibitor Cocktail (Sigma P8340) 5 mL
and cultured cells store at: 2–8 °C Components
The Endoplasmic Reticulum Isolation Kit is used for Calcium chloride, 2.5 M solution 1 mL
GL0010-1KT 1 kit
the efficient enrichment of functional endoplasmic 2.3M Sucrose 25 mL
reticulum (ER) from mammalian tissue or cell Lysosome Isolation Kit Extraction Buffer 5x
culture. The isolated ER are useful for protein OptiPrep™ Dilution Buffer 20 mL
synthesis, degradation and metabolic pathway  sufficient for 25 g (tissue), sufficient for 20 mL Protease Inhibitor Cocktail (Sigma P8340) 5 mL
analysis. Metabolic abnormalities of ER include (packed cells), enrichment of lysosomes from Neutral Red Reagent 1 mL
store at: 2–8 °C
carnitine acetyltransferase deficiency in children, tissues and packed cells
cystic fibrosis, and many more ER protein retention The Lysosome Isolation Kit provides a method LYSISO1-1KT 1 kit
disorders. Suitable for up to 50 g of tissue or for isolating lysosomes from animal tissues and
~20 mL of packed tissue. from cultured cells by differential centrifugation Nuclei Isolation Kit: Nuclei PURE Prep
followed by density gradient centrifugation and/or
 sufficient for 15 nuclei preparations (~1-10×107
• Specially formulated extraction reagents for calcium precipitation. The presence of lysosomes
cells or 1g of tissue per preparation)
research scale applications—save time and can be measured by assaying the activity of the
minimize waste lysosomal marker Acid Phosphatase, with the Centrifugation through a dense sucrose cushion
Acid Phosphatase Assay Kit (Cat. No. CS0740). protects nuclei and strips away cytoplasmic
• Includes a calcium chloride solution—for Separation from other organelles can be measured contaminants. High yield has been obtained from
quick and simple precipitation of rough ER using the appropriate marker detection kits common cell lines (Jurkat, HFN7.1, COS7, HEK293
without need for ultracentrifugation available from Sigma. and MDCK) and tissues (spleen and liver).
For preparation of pure nuclei and fragile nuclei
• Produces functional, intact organelles— from cell lines and solid tissues.
resulting ER are suitable for functional studies,
nuclease and protease........................................................................free
lipid metabolism, and protein profiling
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 23

Components Centrifugation-based Kits Flexibility of Protocol

Nuclei PURE Lysis Buffer 180 mL N C N C Fraction
Nuclei PURE Storage Buffer 90 mL CelLytic™ NuCLEAR™ Extraction Kit
Nuclei PURE Sucrose Cushion Buffer 120 mL
 For mammalian tissue or cultured cells
Nuclei PURE 2 M Sucrose Cushion Solution 475 mL
10% Triton® X-100 Solution 1.7 mL Within this kit is a complete system for preparing Oct-1

store at: 2–8 °C nuclear and cytoplasmic protein extracts from

NUC201-1KT 1 kit
mammalian tissue or cultured cells. All reagents
necessary for extraction are included.
Peroxisome Isolation Kit A number of different procedures in the detailed
technical bulletin enable the selection that best
 isolate peroxisomes from tissues and cells fits a particular application. For example, choose
The Perixosome Isolation Kit provides all the between detergent and non-detergent extraction
necessary reagents and a detailed protocol for of nuclear protein or between the standard
Free Probe
the isolation of highly purified peroxisomes hypotonic lysis buffer for most cell types and
from animal tissues and cells, by differential isotonic lysis buffer for fragile cells. In addition, 1 2 3 4
density gradient centrifugation using iodixanol the kit provides a procedure for salt reduction
[OptiPrep™]. This kit has been used for preparation from the nuclear extract with dilution buffer. Probe: Octamer Motif
of peroxisomes from rat liver, rat kidney and rabbit CelLytic™ NuCLEAR™ offers the flexiblity you need Probe: Oct-1 Motif
liver as well as HEK293 and HepG2 cells. for optimal protein extraction. Extracts can be Preparation Method: IGEPAL® Syringe
Isolated peroxisomes are used for studying prepared in less than 2 hours and are highly pure Figure 2. Gel Shift Assay of Oct-1.
lipid β-oxidation,1 amino acid metabolism2 and since there is little or no cross-contamination Double stranded 32P-labeled Octamer motif oligonucle-
biosynthesis of ether-linked glycerolipids3 and bile between nuclear and cytoplasmic extracts. otide was incubated with either cytoplasmic (C) or nuclear
extracts (N) of HeLa cells prepared using the CelLytic™ Nu-
acids.4,5 1 kit sufficient for 10 extractions (1 ml packed cell CLEAR™ Extraction kit. Two different methods, described
volume) in the protocol were utilized: Method 1 used NP-40
• Specially formulated extraction reagents for 1 kit sufficient for 100 extractions (100 μl packed detergent; Method 2 relied on passages through a syringe
research scale applications - save time and cell volume)
instead of a detergent to extract the nuclei.
minimize waste
Components
Highly Purified Nuclear Fractions
• Produces functional intact organelles - HeLA CHO COS PC-12 Cell
10× Lysis Buffer, Hypotonic 7 mL
3× Dilution and Equilibration Buffer 90 mL
resulting peroxisomes are suitable for functional C N C N C N C N Fraction 1 M DTT 0.4 mL
studies, metabolic assays, protein profiling, and
Extraction Buffer 10 mL
disease state analysis IGEPAL® CA-630 10% Solution 4 mL
5× Lysis Buffer, Isotonic 14 mL
• Compatible with products for structure Oct-1
Protease Inhibitor Cocktail 1 mL
confirmation - easily confirm intactness with store at: −20 °C
companion test kit, Cytochrome C Reductase NXTRACT-1KT 1 kit
Assay Kit (Cat. No. CY0100)
ComponentsPeroxisome Extraction Buffer 5X 100 mL
OptiPrep™ Dilution Buffer 10 mL
OptiPrep™ Density Gradient Medium (Sigma D1556)
100 mL
Protease Inhibitor Cocktail (Sigma P8340) 5 mL
Lit cited: 1. Lazarow, P.B., and De Duve, C., A fatty acyl-CoA oxidizing Free Probe
system in rat liver peroxisomes; enhancement by clofibrate a
hypolipidemic drug. Proc. Natl. Acad. Sci. U. S. A. USA 73, 2043-
2046 (1976); 2. Mannaerts, G.P., and Van Veldhoven, P.P., Metabolic
Figure 1. Purity of Cytoplasmic and Nuclear Proteins.
pathways in mammalian peroxisomes. Biochimie 75, 147-158 (1993);
3. Burdett, K., et al., Peroxisomal localization of Acyl-coenzyme A
The double stranded 32P-labeled Octamer motif oligonu-
reductase (long chain alcohol forming) in guinea pig intestine
mucosal cells. J. Biol. Chem. 266, 12201-12206 (1991); 4. Rodrigues, cleotide was incubated with either cytoplasmic fraction (C)
C.M.P., et al., Formation of delta22-bile acids in rats is not gender or nuclear extract (N) prepared from HeLa, CHO, COS and
specific and occurs in the peroxisomes. J. Lipid Res. 37, 540-550 PC-12 cells using the CelLytic™-NuCLEAR™ extraction kit.
(1996); 5. Une, M., et al., Comparison of side chain oxidation of
potential C27-bile acid intermediates between mitochondria and
peroxisomes of the rat liver: presence of β-oxidation activity for bile
acid biosynthesis in mitochondria. J. Lipid Res. 37, 2550-2556 (1996);
store at: 2–8 °C

PEROX1-1KT 1 kit
24

Mitochondria Isolation Kit Nuclei Isolation Kit: Nuclei EZ Prep Acid phosphatase Assay Kit
The Mitochondria Isolation Kit provides a fast  sufficient for 25 nuclei preparations (~1-10×107  1 kit sufficient for 1,000 assays (multiwell plates),
and easy isolation of an enriched mitochondrial cells/preparation) 1 kit sufficient for 100 assays (tubes)
fraction from animal tissues as well as the testing Provides a high yield of nuclei from commonly Acid phosphatase resides in lysosomes and is a
of the electrochemical proton gradient (ΔΨ) of used mammalian cells, including both adherent marker for the identification of lysosomes in sub-
the inner mitochondrial membrane. Useful for (e.g., HEK293 and COS7) and non-adherent (e.g., cellular fractionations.
mitochondria mediated apoptosis studies. Jurkat and HFN7.1) cell lines and peripheral blood Kit detects acid phosphatase activity in tissues,
mononuclear cells (PBMCs). whole cell extracts, column fractions, and purified
For rapid isolation of nuclei from mammalian cells. enzyme.
94
nuclease and protease........................................................................free Components
48.6
Components p-Nitrophenyl phosphate tablets 5 mg 20 tablets
36.4 Nuclei EZ Lysis Buffer 200 mL Citrate buffer solution, 0.09 M pH 4.8 100 mL
Nuclei EZ Storage Buffer 5 mL p-Nitrophenol Standard solution 10 mM 1 mL
29.8 Acid phosphatase control enzyme 0.2 mL
store at: 2–8 °C
20.6 store at: −20 °C
NUC101-1KT 1 kit
CS0740-1KT 1 kit

Assay Kits for Verification Catalase Assay Kit

6.5 The following kits and reagents are  sufficient for ≥100 tests, enzymatic, determination
of catalase activity in tissues and cells
recommended for determination of
Catalase is a ubiquitous antioxidant enzyme which
Liver mitochondria Heart mitochondria Control organelle presence or integrity after isolation.
2.5 mgP/ml 2.5 mgP/ml cytochrome c catalyses the decomposition of hydrogen peroxide
50 µg/ml Antibodies are used to confirm the presence (H2O2) to water and oxygen. Hydrogen peroxide
Integrity of Isolated Mitchondria of organelle in the lysate. Enzyme activity is formed in the eukaryotic cell as a by-product
Another indication for the integrity of mitochondria is the kits are used to evaluate the integrity of the of various oxidases and superoxide dismutases.
presence of cytochrome c in the intermembrane space Hydrogen peroxide accumulation in cells causes
(between the inner and outer membranes). The presence
organelles.
oxidation of cellular targets such as DNA, proteins,
of free cytochrome c in whole mitochondria can be dem- and lipids leading to mutagenesis and cell death.
onstrated by a Western blot of the preparations produced β-N-Acetylglucosaminidase Assay Kit Removal of the H2O2 from the cell by catalase
using the MITO-ISO kit. Mitochondria from heart show  sufficient for 50 reactions (1 mL), sufficient for
more cytochrome c (at the same total protein concentra-
provides protection against oxidative damage to
500 reactions (100 μL) living cells and its role in oxidative stress related
tion) than mitochondria from liver.
N-acetyl-D-glucosaminidase (NAG) in mammals diseases has been widely studied.
Samples were analyzed by 20% SDS-PAGE and then is a lysosomal enzyme, which takes part in the
blotted. Antibody specific to cytochrome c was then used The Catalase Assay Kit is a simple colorimetric assay
intracellular degradation of glycolipids and for studying catalase activity in various tissues and
in Western blot analysis. Protein concentrations of the
samples used are indicated at the bottom.
glycoproteins. High activities of this enzyme subcellular organelles.
have been detected in human kidney, lung and store at: 2–8 °C
These results show that functional, intact mitochondria liver lysosomes. Elevated levels of serum NAG are
are prepared using the Mitochondria Isolation Kit. associated with certain forms of cancer. CAT100-1KT 1 kit
In addition, the use of this kit together with the
Cytochrome c Oxidase Assay Kit (CYTOC-OX1) facilitates The kit provides the reagents required for Cytochrome c Oxidase Assay Kit
a quick and easy determination of the functionality of a fast and convenient detection of β-N-
both the inner and outer mitochondrial membranes. Acetylglucosaminidase activity in cell lysates, tissue  sufficient for 100 tests, soluble and membrane
homogenates or purified enzyme. It is a useful tool bound mitochondria
 sufficient for 10–20 g (animal tissue), sufficient
for the detection of lysosomes in fractionated cell/ Cytochrome c oxidase [EC 1.9.3.1] is located on
for 50 assays (2 mL), isolation of enriched
mitochondrial fraction from animal tissues tissue samples. the inner mitochondrial membrane dividing the
The kit assay is based on the hydrolysis of NAG mitochondrial matrix from the intermembrane
store at: −20 °C space, and has traditionally been used as a
substrate (NP-GLcNAc) by the enzyme. The
MITOISO1-1KT 1 kit enzymatic hydrolysis of the substrate liberates marker for this membrane. It is also located in the
p-nitrophenylate ion. The reaction product is cytoplasmic membrane of bacteria. Cytochrome
 sufficient for 50 applications (2-5 x 107 cells), c oxidase provides energy for the cell by coupling
isolation of enriched mitochondrial fraction detected colorimetrically at 405 nm.
electron transport through the cytochrome chain
from cells Components with the process of oxidative phosphorylation.
The Mitochondria Isolation Kit provides a fast Dilution Buffer 8 mL
The Cytochrome c Oxidase Assay Kit uses an
and easy method for the isolation of an enriched 4-Nitrophenyl-N-acetyl-β-D-glucosaminide 50 mg
Citrate Buffer Solution, 0.09 M 100 mL optimized colorimetric assay based on observation
mitochondrial fraction from cells.
p-Nitrophenol Standard Solution, 10 mM 1 mL of the decrease in absorbance of ferrocytochrome
store at: −20 °C
β-N-Acetylglucosaminidase from Jack beans 1 vial c measured at 550 nm, which is caused by its
MITOISO2-1KT 1 kit Sodium carbonate 5 g oxidation to ferricytochrome c by cytochrome
store at: 2–8 °C c oxidase. This kit is suitable for the detection of
CS0780-1KT 1 kit mitochondrial outer membrane integrity and
for the detection of mitochondria in subcellular
fractions.
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 25

Components several oxygenases, the most important of which Components

Assay Buffer 5× 25 mL are the cytochrome P450 family of enzymes, Assay Buffer 100 mL
Enzyme Dilution Buffer 2× 20 mL responsible for xenobiotic detoxification. NADPH- Enzyme Dilution Buffer 25 mL
Cytochrome c (Sigma C2506) 50 mg cytochrome c reductase is widely used as an Cytochrome c 50 mg
Cytochrome c positive control 1 vial endoplasmic reticulum marker and as one of the β-Nicotinamide adenine dinucleotide 2'-phosphate
n-Dodecyl β-D-maltoside (Sigma D4641) 10 mg reduced (Sigma N6505) 25 mg
DL-Dithiothreitol solution, 1 M .4 mL
biomarkers for ecological pollution and dietary Cytochrome c Reductase (NADPH) Positive Control (Sigma
store at: −20 °C lipid uptake. C9363) 50 μL
The Cytochrome C Reductase Assay Kit contains Cytochrome c Oxidase Inhibitor Solution 1 mL
CYTOCOX1-1KT 1 kit
all the reagents required for a fast and convenient store at: −20 °C

Cytochrome c Reductase (NADPH) determination of NADPH-Cytochrome C Reductase CY0100-1KT 1 kit

Assay Kit activity in cell and tissue extracts and in purified
microsomes. It has been tested on samples
 1 kit sufficient for 100 tests, determining
prepared from various species of mammalian
cytochrome c reductase activity
tissues such as liver, kidney, brain, spleen, and
Eukaroytic NADPH-cytochrome c reductase heart muscle, as well as on lysates from cell lines
(NADPH-cytochrome P450 reductase, EC1.6.2.4) such as HeLa, HepG2, and Jurkat. In addition, it has
is a flavoprotein localized to the endoplasmic been tested on samples prepared from the yeast
reticulum. It transfers electrons from NADPH to strains P. pastoris and S. cerevisiae.

Antibodies
Species
Product Name   Host Clone No. Form Gene Symbol Reactivity Application Cat. No.
Anti-GM130 (C-terminal)   rabbit - affinity isolated antibody Golga2, rat human IF (i) G7295-200UL
Golga2, mouse mouse IP
GOLGA2, human rat WB CL
Anti-GRP78/BiP (ET-21)   rabbit - IgG fraction of antiserum Hspa5, mouse Xenopus ICC G9043-200UL
HSPA5, human chicken WB
Hspa5, rat hamster
human
mouse
rat
Anti-Nuclear Pore Complex mouse 414 purified immunoglobulin - Xenopus EM N8786-200UL
Proteins   human ICC
mouse IP
rat WB
yeast
Anti-PMP70   rabbit - affinity isolated antibody Abcd3, mouse human IF (i) P0497-200UL
ABCD3, human mouse IP
Abcd3, rat rat WB CL

Application Abbreviation Table

Application Abbreviation Application Abbreviation
ANA-indirect immunofluorescence IF (ANA) Immunohistochemistry IHC
Capture ELISA ELISA (c) Immunohistochemistry (formalin-fixed, IHC (p)
Direct ELISA ELISA (d) paraffin-embedded sections)
Direct immunofluorescence IF (d) Immunohistochemistry (frozen sections) IHC (f )
Dot blot DB Immunoprecipitation IP
Dot immunobinding DIBA Indirect ELISA ELISA (i)
Electron microscopy EM Indirect immunofluorescence IF (i)
Enzyme immunoassay EIA Microarray ARR
Flow cytometry FACS Neutralization Neutral
Immunoblotting WB Ouchterlony double diffusion ODD
Immunoblotting (chemiluminescent) WB CL Particle immunofluorescence PIFA
Immunocytochemistry ICC Quantitative precipitin assay QPA
Immunoelectrophoresis IEP Radioimmunoassay RIA
26

ECACC® Cell Lines

ECACC® Cell Lines

Sigma Life Science has over 1500 ECACC® reduced. All cell lines are authenticated and and toxicology. For the comprehensive and
cell lines available in Europe and the mycoplasma tested so you can use them in up-to-date list of ECACC® cell lines Sigma
United States. Many hard-to-get cell lines your applications with confidence. The cell provides, visit sigma.com/ecacc.
are included so that the time and effort lines can be used in a variety of applications,
required for procurement will be greatly including cancer research, neurobiology,

Name Description Biological Source Cat. No.

A2780 Cell Line ovarian carcinoma human 93112519-1VL
A2780cis Cell Line ovarian carcinoma human 93112517-1VL
A549 Cell Line human lung carcinoma human 86012804-1VL
B9 Cell Line Mouse B cell hybridoma from mouse 96080128-1VL
C2C12 Cell Line C3H muscle myoblast from mouse 91031101-1VL
CACO-2 Cell Line Caucasian colon adenocarcinoma human 86010202-1VL
1301 Cell Line T-cell leukemia human 01051619-1VL
1321N1 Cell Line Glial cells from brain astrocytoma human 86030402-1VL
293 Cell Line embryonic kidney human 85120602-1VL
CHO-K1 Cell Line ovary from hamster 85051005-1VL
HeLa Cell Line epitheloid cervix carcinoma human 93021013-1VL
Hep G2 Cell Line hepatocyte carcinoma human 85011430-1VL
Jurkat E6.1 Cell Line leukemic T cell lymphoblast human 88042803-1VL
L929 Cell Line mouse C3H/An connective tisue from mouse 85011425-1VL
MCF7 Cell Line breast adenocarcinoma human 86012803-1VL
MDA-MB-231 Cell Line human breast adenocarcinoma human 92020424-1VL
MRC-5 pd13 - - 05011802-1VL
MRC-5 PD 19 Cell Line Human fetal lung human 05072101-1VL
Nb2-11 Cell Line Thymus/lymph node, lymphoblast morphology from rat 97041101-1VL
NIH 3T3 Cell Line Embryonic fibroblast murine 93061524-1VL
OE33 Cell Line Caucasian esophageal carcinoma human 96070808-1VL
U-2 OS Cell Line osteosarcoma human 92022711-1VL
RAW 264.7 Cell Line Macrophage from blood murine 91062702-1VL
SH-SY5Y Cell Line Neuroblast from neural tissue. human 94030304-1VL
SP2/O-Ag14 Cell Line Non-producing hybridoma murine 85072401-1VL
THP 1 Cell Line Leukemic monocyte human 88081201-1VL
tsa201 Cell Line embryonal kidney, SV40 transformed human 96121229-1VL
U937 Cell Line Lymphoblast from lung human 85011440-1VL
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 27

Centrifugation Equipment

Centrifugation Equipment
Corning® Microcentrifuges
Corning® LSE™ high speed microcentrifuge
Easy-to-use, digital control interface and high-speed performance for quick nucleic acid and protein separations. Has two control knobs to precisely set rotor
speed and run time, and a “Speed/G-Force” knob to toggle the display between rpm and calculated g-force. Incorporates a brushless drive and unique air
cooling system to both reduce noise and minimize sample heating even during prolonged runs at maximum speed.

• Fast acceleration to a maximum speed 13,300 rpm/16,300 × g

• Complete with easy access, 24 × 1.5/2.0 mL rotor
CE compliant.............................. Yes

Mfr Designation AC Cat. No.

Corning® No.6766-HS 230 V (Schuko plug) CLS6766HS-1EA
Corning® No.6767-HS 230 V (UK plug) CLS6767HS-1EA
Corning® No.6765HS 120 V CLS6765HS-1EA

Corning® LSE™ mini microcentrifuge

Designed for quick spin downs of micro-samples, the operation is simple and convenient. After loading sample tubes, close the lid and the rotor accelerates
quickly to 6000 rpm (2000 × g). This speed range is ideal for bringing small droplets to the bottom of the tubes, for micro-filtrations, or basic separations.
Includes an 8-place rotor that will accept standard 1.5 mL to 2.0 mL microcentrifuge tubes, and adapters are included for 0.5/0.4 mL and 0.2 mL tubes. Also
included is a 4-place rotor for PCR strip tubes.
CE compliant.............................. Yes

Mfr Designation AC Cat. No.

Corning® No.6766 230 V (Schuko plug) CLS6766-1EA
Corning® No.6767 230 V (UK plug) CLS6767-1EA
Corning® No.6765 120 V CLS6765-1EA
28

Hettich® Centrifuges
Hettich® EBA 20 Centrifuge
Practical and handy, the EBA 20 is ideal for a small quantity of samples. This small,
microprocessor-controlled centrifuge is supplied with an 8-place fixed angle rotor. It accepts
a variety of tubes up to a volume of 15 mL and generates an RCF of 3,461. This centrifuge is
supplied with an 8-place fixed angle rotor.
• Small, economy benchtop centrifuge
• Non-refrigerated
• Capacity: 8 × 15 mL
• Max RPM/RCF: 6,000/3,421(Fixed Angle)
• Dimensions (H×W×D): 216 mm (8.5 in.) × 231 mm (9.0 in.) × 292 mm (11.5 in.)
• One multi-functional 8-place angled rotor included supporting: blood tubes up to 10 mL,
round bottom glass & plastic tubes up to 15 mL; conical tubes up to 15mL
• Weight: 4 kg (8.75 lbs)
• CE Compliant
Product Mfg. No. AC Input Cooling Cat. No.
EBA 20 Hettich® 2002-01 115 V No Z601039-1EA

Hettich® EBA 21 Centrifuge

Maximum performance in microliter centrifugation coupled with reliability for routine
laboratory tasks. The EBA 21 is equipped with a variable, maintenance-free frequency drive
that generates a max RCF of 23,907. With its' wide range of rotors (rotor not included), the EBA
21 can easily meet all the demands placed on a small, high-performance centrifuge.
• Basic economy benchtop centrifuge
• Non-refrigerated
• High performance
• Capacity: 6 × 50 mL
• Max RPM/RCF: 18,000/23,907 (Fixed Angle)
• Dimensions (H×W×D): 247 mm (9.75 in.) × 275 mm (10.75 in.) × 330 mm (13.0 in.)
• Multiple rotors available supporting: microliter tubes (biocontained), round bottom tubes up
to 50 mL, conical tubes up to 50 mL; blood tubes up to 10 mL
• Compact with a small footprint
• Weight: 11 kg (24.25 lbs)
• CE Compliant
Rotors and other centrifuge accessories are sold separately

Product Mfg. No. AC Input Cooling Cat. No.

EBA 21 Hettich® 1004-01 115 V No Z601136-1EA
 Order sigma.com/order Technical service sigma.com/techinfo sigma.com/lifescience 29

MIKRO 200/200R Centrifuges

The MIKRO 200 microliter centrifuges are not only fast, but the MIKRO 200R refrigerates
quickly: down to + 4 °C in 10–15 minutes with the Fast Cool function. Highly reliable
refrigeration ensures that even thermosensitive samples are centrifuged gently. Rotors sold
separately.
• Standard micro centrifuge
• Available in non-refrigerated (200) and refrigerated (200R) versions
• Capacity: 24 × 1.5/2.0 mL
• Max RPM/RCF: 14,000/18,626 (Fixed Angle)
• Choice of 24 or 30 place rotors, PCR strip rotor, or Cryo tube rotor
• Bio-containment, autoclavable; phenol resistant lids available
• Fast cool function: down to +4 °C in 10-15 minutes
• CE Compliant
Rotors and other centrifuge accessories are sold separately.

Product Mfg. No. AC Input Cooling Cat. No.

MIKRO 200 Hettich® 2400-01 115 V No Z652105-1EA
MIKRO 200 Hettich® 2400 240 V No Z652091-1EA
MIKRO 200R Hettich® 2405-01 115 V Yes Z652121-1EA

ROTANTA 460/460R Centrifuges

Enhanced performance and an extended range of accessories guarantee fast, trouble-free
operation in daily laboratory use. With a maximum capacity of 3 liters per run, the ROTANTA
460 centrifuges are proving themselves in clinics and laboratory facilities, as well as in
biotechnology and life science departments. Rotors sold separately.
• Large capacity universal benchtop centrifuge
• Available in non-refrigerated (460) and refrigerated (460R) versions
• Temperature Range (460R): –20 °C to + 4 °C
• Capacity: 4 × 750 mL (Swing-out)
• Max RPM/RCF: 15,000/24,400 (Fixed Angle)
• 89 user defined programs
• High speed plate rotor (6,446 g)
• Special braking ramp for spinning blood bags
• Bi-Directional serial interface (optional)
• Control Panel lock with key (optional)
• CE Compliant
Speciality applications available:
• Blood bags
• Cytology
• Chrome bath and Schlenk tubes
Rotors and other centrifuge accessories are sold separately.

Product Mfg. No. AC Input Cooling Cat. No.

ROTANTA 460 Hettich® No.5600-01 115 V No Z623520-1EA
ROTANTA 460 Hettich® No. 5600 240 V No Z623512-1EA
ROTANTA 460R Hettich® No.5605-01 115 V Yes Z623547-1EA
ROTANTA 460R Hettich® No.5605 240 V Yes Z623539-1EA
30

ROTINA 420/420R Centrifuges

The ROTINA 420 is a compact benchtop centrifuge that is well equipped for all centrifuge
applications. This centrifuge has been conceived for high sample throughput as well as for
high sample volumes. With a maximum capacity of 4 × 600 mL, 140 blood collection tubes or
16 microtiter plates they are ideal centrifuges for applications in clinical chemistry as well as
biotechnology and life science research facilities. Rotors sold separately.
• Available in non-refrigerated (420) and refrigerated (420R) versions
• Maximum capacity of: 4 × 600 mL, 52 × 15 mL conical tubes, 84 blood/urine tubes or
16 microtiter plates
• Noise Dampening Technology
• Self diagnostic testing
• 98 user defined programs
Rotors and other centrifuge accessories are sold separately.
Product Mfg. No. AC Input Cooling Cat. No.
ROTINA 420 Hettich® No.4701-01 115 V No Z723428-1EA
ROTINA 420 Hettich® No.4701 240 V No Z723525-1EA
ROTINA 420R Hettich® No.4706-01 115 V Yes Z723630-1EA
ROTINA 420R Hettich® No.4706 240 V Yes Z723754-1EA

ROTOFIX Centrifuge
The ROTOFIX 32A is ideally equipped for tasks in clinical chemistry and cytology. However, this
versatile centrifuge can handle a lot more than just medical applications. With its' wide variety
of accessories, the ROTOFIX 32A can also be used for the preparation of samples in industrial
and research laboratories. Rotors sold separately.
• Economy universal benchtop centrifuge
• Non-refrigerated
• Capacity: 4 × 100 mL
• Max RPM/RCF: 6,000/4,186 (Swing-out)
• Dimensions (H×W×D): 257 mm (10.0 in.) × 366 mm (14.5 in.) × 430 mm (17.0 in.)
• Rugged, versatile and indispensable for routine laboratory tasks
• Wide variety of accessories
• Exchangeable rotors available support: round bottom tubes up to 100 mL, conical tubes up
to 50 mL; cytology up to 8 slides carriers
• Weight: 23 kg (50.5 lbs)
• CE Compliant
Rotors and other centrifuge accessories are sold separately.

Product Mfg. No. AC Input Cooling Cat. No.

ROTOFIX 32A Hettich® 1206-01 115 V No Z601446-1EA
 Biointegrity
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media, sera and reagents for over 25 years.

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That’s why we offer an unparalleled
line of cell culture solutions. From
classical and specialty media, to
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exactly what you’d expect from a
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For more information on

how our cell culture offering
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