Instructions 28-4039-56 AE Cell Preparation Media

Ficoll-Paque™ PREMIUM
Ficoll-Paque PREMIUM 1.084
Ficoll-Paque PREMIUM 1.073
Intended use
For in vitro isolation of mononuclear cells and/or granulocytes from
peripheral blood, bone marrow and umbilical cord blood.
The products are intended for research use only, and shall not be used
in any clinical or in vitro procedures for diagnostic purposes.
Safety
For use and handling of the products in a safe way, please refer to the
Safety Data Sheet.
Ficoll-Paque PREMIUM products contain
100 ml and 500 ml of sterile Ficoll-Paque PREMIUM product containing
Ficoll PM400, sodium diatrizoate and edetate calcium disodium in water
for injection (WFI). The product is sterile, manufactured under a Quality
Management System certified to ISO 13485 and has low levels of endo-
toxin (< 0.12 EU/ml).
Table of Contents
1 Introduction ................................................................................................. 3
2 Advice on handling ................................................................................... 5
3 Procedure ..................................................................................................... 8
4 Ordering information .............................................................................. 14
5 References ................................................................................................... 15

2 28-4039-56 AE
1 Introduction
In 1968, Bøyum described a method using low viscosity Ficoll™ and
sodium metrizoate, of the proper density and osmotic strength, to
isolate mononuclear cells (1). Sodium metrizoate has been success-
fully substituted with sodium diatrizoate by numerous workers (2,3).
The method described in Section 3 for isolating mononuclear cells
has been used with human peripheral blood and umbilical cord blood
samples using Ficoll-Paque PLUS/PREMIUM products. It has also
been used to prepare mononuclear cells from bone marrow (4,5).
The procedure to isolate granulocytes is based on customers' expe-
riences.

Principle of the procedure

Defibrinated or anticoagulant-treated blood is layered on the Ficoll-
Paque PREMIUM solution and centrifuged for a short period of time.
Differential migration during centrifugation results in the formation
of layers containing different cell types:
• The bottom layer contains erythrocytes, which have been aggre-
gated by Ficoll PM400 and therefore sediment completely in the
Ficoll-Paque PREMIUM layer.
• The layer immediately above the erythrocyte layer contains
mostly granulocytes, which at the osmotic pressure of the Ficoll-
Paque PREMIUM solution, attains a density great enough to
migrate through the Ficoll-Paque PREMIUM layer.
• At the interface between the plasma and the Ficoll-Paque
PREMIUM layer, mononuclear cells are found together with other
slowly sedimenting particles (e.g. platelets) with low density.
Mononuclear cells are then recovered from the interface and
subjected to short washing steps with a balanced salt solution
to remove platelets, density gradient medium and plasma.

28-4039-56 AE 3
Ficoll-Paque PREMIUM products
Ficoll-Paque PREMIUM products are sterile Ficoll PM400/sodium dia-
trizoate solutions. They have the proper density, viscosity and osmotic
pressure for use in a simple and rapid cell separation procedure of
blood and bone marrow.
Ficoll-Paque PREMIUM products are manufactured under a Quality
Management System certified to ISO 13485. All Ficoll-Paque products
also provide the additional advantage of low levels of endotoxin.
Ficoll-Paque PREMIUM, with the density of 1.077 g/ml, is based on
Ficoll-Paque PLUS, which has a proven track record as a sterile
density medium for the isolation of high yields of mononuclear cells
from human peripheral blood, bone marrow and umbilical cord
blood.
Ficoll-Paque PREMIUM 1.084 and Ficoll-Paque PREMIUM 1.073, have
densities of 1.084 and 1.073 g/ml, respectively. These may be used
when higher or lower densities than the standard 1.077 g/ml are
required.
Ficoll-Paque PREMIUM 1,084 can be used for isolating higher-density
human mononuclear cells. It can also be used for separating blood
cells from mice and rats. The reason is that the lymphocytes in ro-
dents have a slightly higher average density than in humans (6,7).
As a result, a fraction of the rodent lymphocytes will move to the
bottom of a 1.077 g/ml density gradient medium during centrifuga-
tion, contaminating the granulocyte layer and decreasing the
mononuclear cell recovery.
Ficoll-Paque PREMIUM 1.073 can be used when isolating lower
density mononuclear cells, for example mesenchymal stromal cells
or monocytes. The higher density lymphocytes and granulocytes will
sediment through Ficoll-Paque PREMIUM 1.073 to the bottom of the
tube, thereby enriching the lower density cells at the interface.
For an overview of the Ficoll-Paque PREMIUM applications, refer to
the following table:

4 28-4039-56 AE
 Ficoll-Paque Species Tissue Cells
PREMIUM
1.073 g/ml human • blood • mononuclear cells
of lower density
• bone marrow, etc. • mesenchymal
stromal cells
1.077 g/ml human • peripheral blood • mononuclear cells
• bone marrow
• umbilical cord
blood
1.084 g/ml human • peripheral blood • mononuclear cells
• bone marrow of higher density
• umbilical cord
blood
mice and rat • blood • lymphocytes

2 Advice on handling
Precautions
Upon contact with biological materials, all reagents and equipment
should be treated as potentially biohazardous. Practice Universal
Precautions. All waste should be considered biohazaradous , and
disposed in accordance with your institutions procedures.
All glass has the potential for breakage; precautionary measures
should be taken during handling.
Precautions should be taken to prevent injury when pulling off the
metal seal.

Aseptic procedures
Use aseptic procedures at all times as Ficoll-Paque PREMIUM prod-
ucts do not contain antibiotics or preservatives.

28-4039-56 AE 5
Storage
Ficoll-Paque PREMIUM products are stable for 3 years if stored un-
opened between 4°C and 30°C and protected from direct light. Please
contact GE Healthcare for internal references.

Indications of instability
Deterioration of the Ficoll-Paque PREMIUM products is indicated by
the appearance of a distinct yellow color or particulate material in
the clear solution.

Expected results
Typical expected results when isolating mononuclear cells from fresh
human peripheral blood (approximately 2 hours old) with Ficoll-Paque
PREMIUM.
Mononuclear cells
• 95 ± 5% of cells in isolate are mononuclear cells.
• 95 ± 5% viability 1
• 60 ± 20% recovery of mononuclear cells from the original blood 2
sample.
1 Mononuclear cell viability was determined by the Trypan blue exclusion test (8).
2 The white blood cell count on the starting blood sample was done in a hemacytometer
(9). A differential count of the white blood cells was then performed to determine the
amount of granulocytes in the starting blood sample.

Other cells
• Max. 5% granulocytes 1 , max. 10% erythrocytes of cells in isolate.
1 The differential cell count was obtained from a smear of the lymphocyte fraction treated
with Wright’s Stain (9)

6 28-4039-56 AE
Factors affecting the isolation of mononuclear cells
Age of blood
The blood should be as fresh as possible and free of clots. Delays in
processing the blood can result in loss of viability, lower cell recover-
ies and more contaminating granulocytes and/or erythrocytes.
Blood volume
The blood volume and tube diameter are factors determining the
height of the blood sample in the tube and, consequently, the cen-
trifugation time. Increasing the height of the blood sample in the
tube increases erythrocyte contamination. The separation, however,
is not appreciably affected by the diameter of the tube. As a result,
a larger volume can be separated in a tube of larger diameter, chosen
so that the height of the blood sample in the tube and the separation
time are constant.
Yield and purity
The yield and the degree of purity of the mononuclear cells depend
on the efficiency of erythrocyte removal. When erythrocytes in whole
blood are aggregated, some mononuclear cells are trapped in the
clumps and, therefore, sediment with the erythrocytes. This tendency
is reduced by diluting the blood.
A temperature of 18°C gives optimum results. Aggregation of erythro-
cytes is increased at higher temperatures (37°C) which decreases
yield, but at low temperatures (4°C) the rate of aggregation is de-
creased, increasing the time of separation. Increasing the centrifu-
gation time with 5 to 10 minutes may help reducing erythrocyte
contaminations.
Platelet contamination
If it is important to remove all platelets from the mononuclear cell
fraction a second centrifugation in a 4 to 20% sucrose gradient lay-
ered over Ficoll-Paque PREMIUM can be applied. This procedure will
effectively remove any platelet contamination (10). Platelets will re-
main at the top of the sucrose gradient and mononuclear cells will
sediment through the sucrose gradient to the top of the Ficoll-Paque
PREMIUM layer.

28-4039-56 AE 7
3 Procedure
3.1 Materials
Materials required but not provided
• Sterile balanced salt solution or other standard salt solutions.
See Section Preparation of reagents.
• Centrifuge with swing-out rotor.
• Sterile tubes and pipettes.
• Sterile needles and syringes.
• Red blood cell lysis solution of choice (if isolating granulocytes).

Sample volume
Sample volume (4 ml total)
Mix 2 ml defibrinated or anticoagulant-treated blood with 2 ml sterile
salt solution.
Larger blood volumes
Larger volumes of blood may also be processed with the same
efficiency of separation. This is achieved by increasing the diameter
of the centrifuge tube while maintaining approximately the same
height of the Ficoll-Paque PREMIUM product (approximately 2.4 cm)
and of blood sample (approximately 3.0 cm) in the centrifuge tube
(11).
Smaller blood volumes
Smaller quantities of blood can be processed rapidly by a
modification of the recommended procedure (12).

8 28-4039-56 AE
Preparation of reagents
Use of reagents
This procedure describes the isolation of mononuclear cells and
granulocytes using Ficoll-Paque PREMIUM and the balanced salt
solution described below as a diluent and washing solution. Other
diluents and washing fluids such as isotonic Ca2+/Mg2+ free phos-
phate buffered saline (e.g. Dulbecco's PBS), salt solutions (e.g. Hank's)
or cell culture media (e.g. RPMI 1640) may also be used. The same
procedure is recommended when separating cells using Ficoll-Paque
PREMIUM 1.084 or Ficoll-Paque PREMIUM 1.073.
Ficoll-Paque PREMIUM product
Warm the Ficoll-Paque PREMIUM solution to 18°C to 22°C before
use.
Balanced salt solution
It is recommended to use commercial available ready-to-use sterile
salt solutions.
To prepare a laboratory prepared balanced salt solution, mix 1 vol-
ume stock solution A with 9 volumes stock solution B.
Note: It is important to sterilize the prepared balanced salt solution.
At least 20 ml for each sample should be processed.
Stock solution A Conc. (g/l).
Anhydrous D-glucose 0.1% 1.0
CaCl2 × 2H2O 5.0 × 10-5 M 0.0074
MgCl2 × 6H2O 9.8 × 10-4 M 0.1992
KCl 5.4 × 10-3 M 0.4026
Tris 0.145 M 17.565
Conc. HCl 10 N to pH 7.6
Distilled water 1 to 1000 ml
1 Dissolve in approximately 950 ml distilled water and add 10 N HCl until pH is 7.6 before
adjusting the volume to 1 l.

28-4039-56 AE 9
 Stock solution B Conc. (g/l)
NaCl 0.14 M 8.19

3.2 Procedure for isolation of mononuclear cells

Specimen collection and handling
Fresh blood should be used to ensure high recovery, purity and via-
bility of the isolated mononuclear cell fractions. Prepare sample at
18°C to 20°C as follows:

Step Action
1 To a 10 to 15 ml centrifuge tube add 2 ml of defibrinated or
anticoagulant-treated blood 1 and an equal volume of bal-
anced salt solution (final volume 4 ml).
2 Mix the blood and buffer by inverting the tube several times
or by drawing the mixture in and out of a pipette.
1 Anticoagulants: Heparin, EDTA, citrate, acid citrate dextrose (ACD)
and citrate phosphate dextrose (CPD) can be used. Defibrinated
blood requires no anticoagulant.

10 28-4039-56 AE
Density gradient separation
Step Action
3 Invert the Ficoll-Paque PREMIUM bottle several times to en-
sure thorough mixing.
• For withdrawal of Ficoll-Paque PREMIUM by syringe:

- Snap-off the polypropylene cap to expose the centres

of the rubber stopper.
- Disinfect the stoppers with an alcohol swab by rubbing
the stopper firmly for several seconds and allow them
to dry prior to use.
- Insert syringe needle through the septum.

- Invert the bottle and withdraw the required volume of

Ficoll-Paque PREMIUM.
• For withdrawal of Ficoll-Paque PREMIUM by pipette:

- Remove the snap-off polypropylene cap. Lift the alu-

minum ring. Pull off the metal seal. Remove the silver
ring. Remove the rubber septum. Using aseptic tech-
niques, withdraw the required volume of Ficoll-Paque
PREMIUM.
Note:
The content of the bottle is no longer sterile and the bottle
should be disposed to waste.
4 Add Ficoll-Paque PREMIUM (3 ml) to the centrifuge tube.

28-4039-56 AE 11
 Step Action
5 Carefully layer the diluted blood sample (4 ml) on Ficoll-Paque
PREMIUM.

Blood sample

Ficoll-Paque PREMIUM

Note:
When layering the sample do not mix Ficoll-Paque PREMIUM
and the diluted blood sample.
6 Centrifuge at 400 × g for 30 to 40 min at 18°C to 20°C.
7 Draw off the upper layer containing plasma and platelets
using a sterile pipette, leaving the layer of mononuclear cells
undisturbed at the interface.

Upper layer removed

Plasma and platelets

Mononuclear cells Mononuclear cells

Ficoll-Paque PREMIUM
Granulocytes, erythrocytes

Note:
Care should be taken not to disturb the layer of mononuclear
cells. It is also possible to withdraw the mononuclear cell
layer with a pipette without first removing the upper plasma
layer. The upper layer of plasma, which is essentially free of
cells, may be saved for later use.

12 28-4039-56 AE
 Step Action
8 Transfer the layer of mononuclear cells to a sterile centrifuge
tube using a sterile pipette.
Note:
It is critical to remove all of the interface but a minimal
amount of Ficoll-Paque PREMIUM and supernatant. Removing
excess Ficoll-Paque PREMIUM causes granulocyte contami-
nation, removing excess supernatant results in unnecessary
contamination by platelets and plasma proteins.

Washing the cell isolate

Step Action
9 Estimate the volume of the transferred mononuclear cells.
Add at least 3 volumes (approximately 6 ml) of balanced salt
solution to the mononuclear cells in the centrifuge tube.
10 Suspend the cells by gently drawing them in and out of a
pipette.
11 Centrifuge at 400 to 500 × g for 10 to 15 min at 18°C to 20°C.
Note:
A centrifugation at high speed increases the mononuclear
cell recovery. However, if it is important to also get rid of
platelets a lower centrifugation speed is recommended (60
to 100 × g).
12 Remove the supernatant.
13 Resuspend the mononuclear cells in 6 to 8 ml balanced salt
solution.
14 Centrifuge at 400 to 500 × g (or 60 to 100 × g for removal of
platelets) for 10 min at 18°C to 20°C.
15 Remove the supernatant.
16 Resuspend the cell pellet in a medium appropriate for the
application.

28-4039-56 AE 13
3.3 Procedure for isolation of granulocytes
Step Action
1 Perform Ficoll-Paque gradient centrifugation as described
above in Section 3.2 Procedure for isolation of mononuclear
cells step 1 to 8.
2 Draw off the upper layer of Ficoll-Paque PREMIUM using a
sterile pipette, leaving the layer of granulocytes undisturbed.
3 Collect the thin white cell layer of granulocytes above the
red blood pellet with a pipette and transfer to a sterile 50 ml
centrifuge tube.
4 Resuspend the cells in at least five volumes of balanced salt
solution and centrifuge at 400 × g for 15 minutes.
5 Lyse remaining red blood cells with any red blood cell lysis
solution of choice.
6 Centrifuge the granulocytes at 400 to 500 × g for 10 to 15
minutes at 18°C to 20°C.
7 Remove the supernatant.
8 Resuspend the granulocytes in 6 to 8 ml balanced salt solu-
tion.
9 Centrifuge at 400 to 500 × g for 10 min at 18°C to 20°C.
10 Remove the supernatant.
11 Resuspend the cell pellet in the medium appropriate for the
application.

4 Ordering information
Product Pack size Code No
Ficoll-Paque PREMIUM 6 × 100 ml 17-5442-02
Ficoll-Paque PREMIUM 6 × 500 ml 17-5442-03
Ficoll-Paque PREMIUM 1.084 6 × 100 ml 17-5446-02
Ficoll-Paque PREMIUM 1.073 6 × 100 ml 17-5446-52

14 28-4039-56 AE
5 References
1 Bøyum, A. Isolation of mononuclear cells and granulocytes from
human blood. Scand. J. Clin. Lab. Invest. 21, Suppl. 97 (Paper IV),
77–89 (1968).
2 Bain, B. and Pshyk, K. Enhanced reactivity in mixed leukocyte
cultures after separation of mononuclear cell on Ficoll-Hypaque.
Transplantation Proceedings 4, 163–164 (1972).
3 Fotino, M. et al. Instant lymphocytes. Vox Sang 21, 469–470
(1971).
4 Arkin, S. et al. Expression of intercellular adhesion molecule-1
(CD54) on hematopoietic progenitors. Blood 77, 948 (1991).
5 Deguchi, Y. and Kehrl, J. H. Selective expression of two homeobox
genes in CD34-positive cells from human bone marrow. Blood
78, 323 (1991).
6 Leene, W. et al. Lymphocyte differentiation in the rabbit thymus.
Ann Immunol (Paris) 127, 911-921 (1976).
7 Bøyum, A et al. Scand J Immunol. Separation of leucocytes: im-
proved cell purity by fine adjustments of gradient medium den-
sity and osmolality.34:697-712. (1991)
8 Phillips, H. J. Dye exclusion tests for cell viability in Tissue Culture:
Methods and Applications (Kruse, P. F. Jr. and Patterson, M. J. Jr.
eds.), Academic Press, pp 406–408 (1973)
9 Brown, L. in Hematology: Principles and Procedures, Lea and
Febinger, Philadelphia, USA. (1973).
10 Perper, R. J. et al. Purification of lymphocytes and platelets by
gradient centrifugation. J. Lab. and Clin. Med. 72, 842–848, (1968).
11 Skoog, W. A. and Beck, W. S. Studies in the fibrinogen, dextran
and phytohemagglutinin methods of isolating leukocytes. Blood
11, 436–454 (1956).
12 Bøyum, A. Separation of white blood cells. Nature 204, 793–94
(1964).

28-4039-56 AE 15
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www.gelifesciences.com/contact
GE Healthcare Bio-Sciences AB
Björkgatan 30
751 84 Uppsala
Sweden
www.gelifesciences.com/cellprep

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Ficoll and Ficoll-Paque are trademarks of GE Healthcare companies.
© 2005-2013 General Electric Company – All rights reserved.
First published Oct. 2005
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