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Leucosep ®
Enrichment of lymphocytes from whole blood
Leucosep ® is designed to maximize the effectiveness of lymphocyte or mononuclear cell separation from whole
blood and bone marrow. The single greatest variability in peripheral blood mononuclear cell (PBMC) separations is
the interface between the anticoagulated blood or bone marrow and the lymphocyte separation media. Maintaining
this discontinuous gradient while loading your sample is critical to centrifugal separations using a barrier protocol.
Using Greiner Bio-One’s Leucosep ® tube will maintain that discontinuous gradient and improve your PBMC
separations.
The Leucosep ® tube consists of a high quality GREINER polypropylene centrifuge tube which contains a porous
barrier secured at a fixed point in the tube. The porous barrier consists of high quality inert polyethylene and offers a
pore size that perfectly matches its intended applications. This porous barrier eliminates the time consuming and
troublesome sample overlay for filling and prevents the recontamination of the enriched cell fraction with red blood
cells.
Leucosep ® tubes allow a choice of media, whether Lymphoprep ® , NycoPrep 1.077 ® or another separation medium
is preferred. Greiner Bio-One offers a complete selection of Leucosep ® tubes to meet your small or large sample
processing requirements with 10 mL and 50 mL sizes.
Cat. No. Description Tube Size Sample Volume
F e a t u r e s
� Simplified filling due to porous barrier
� Performance in about 15 minutes at RT
� No recontamination with red blood cells
� No special lab equipment required
� Thinning of red blood cells & granulocytes
� No blocking of marker molecules
� Enrichment directly from whole blood
Quantity per
Pack / Case
163290 Leucosep ® tube, sterile 10 mL 3-8 mL blood 50 / 500
163290P Leucosep ® tube, sterile 10 mL 3-8 mL blood 50 / 50
227290 Leucosep ® tube, sterile 50 mL 15-30 mL blood 25 / 300
227290P Leucosep ® tube, sterile 50 mL 15-30 mL blood 25 / 25
Greiner Bio-One North America, Inc. Toll Free: 800.884.4703
4238 Capital Drive Tel: 704.261.7800
Monroe, NC 28110 Fax: 704.261.7899
www.gbo.com/bioscience E-Mail: info@us.gbo.com GBOI/NC/3000/V6/05/07

Instruction Manual
Leucosep ® (163290, 163290P, 227290, 227290P)
The Method
Leucosep ® has been developed for the optimal separation of lymphocytes and peripheral blood mononuclear cells (PBMC’s) from
human whole blood and bone marrow by density gradient centrifugation. Anticoagulated blood or bone marrow can be poured directly
from the blood sampling tube into the Leucosep ® tube. The porous barrier prevents mixture of the sample material with the separation
medium. During centrifugation, lymphocytes and PBMC’s are separated from unwanted erythrocytes and granulocytes on the basis of
their buoyant density and enriched in an interphase above the separation medium. When separation is complete, the barrier prevents
recontamination of the enriched cell fraction during harvest
Preparation
� Warm-up separation medium (your choice of brands of lymphocyte separation medium, such as Lymphoprep or NycoPrep 1.077) to
room temperature (RT) protected from light.
� Fill the Leucosep ® tube with the separation medium: 3 mL when using tubes 163290 or 163290P; 15 mL when using tubes 227290 or
227290P.
� Close the tubes containing the separation medium with the screw-cap and centrifuge for 30 seconds at 1000 x g at RT. The separation
medium is now located below the porous barrier.
� The tubes are now ready for filling with anticoagulated blood or bone marrow aspirate. Dilution of the sample material with a balanced
salt solution is not implicitly necessary, but it can help to improve the result of the separation. For blood a dilution ratio of 1:2, for bone
marrow a ratio of 1:4 is recommended.
Procedure
Filling with sample material Before centrifugation After centrifugation Harvest with a Pasteur pipette or by decanting
into another centrifuge tube
1) Pour the anticoagulated sample material (blood or bone marrow aspirate, diluted with balanced salt solution if necessary) directly from
the blood sampling tube carefully into the Leucosep® tube: 3-8 mL of sample material when using 163290 or 163290P; 15-30 mL of
sample material when using tubes 227290 or 227290P.
2) Centrifuge 10 minutes at 1000 x g at RT or 15 minutes at 800 x g at RT in a swinging bucket rotor. Switch-off brakes of the centrifuge.
3) After centrifugation, the sequence of layers occurs as follows (seen from top to bottom):
a) Plasma
b) Enriched fraction (interphase of lymphocytes/PBMC’s)
c) Separation medium
d) Porous barrier
e) Separation medium
f) Pellet (erythrocytes and granulocytes)
Collection and discarding of the plasma layer fraction up to a minimum remnant of 5 to 10 mm above the interphase helps to prevent
contamination of the enriched cells with platelets.
4) Harvest the enriched cell fraction (lymphocytes / PBMC’s) by means of a Pasteur pipette or by pouring the supernatant above the
porous barrier from the Leucosep ® tube into another centrifuge tube. The porous barrier effectively avoids recontamination with pelleted
erythrocytes and granulocytes.
5) Wash the enriched cell fraction (lymphocytes / PBMC’s) with 10 mL of phosphate-buffered saline (PBS), subsequently centrifuge for 10
minutes at 250 x g.
6) Repeat washing step twice; resuspend the cell pellet with 5 mL of PBS.
Caution
Handle all biological samples and blood collection lancets, needles and blood collection sets in accordance with the policies and procedures of your
facility. In case of exposure or contamination with blood or other biological samples (e.g. accidental puncture injury) initiate appropriate medical
treatment as such material has to be considered potentially infective with HBV, HCV (hepatitis), HIV (AIDS) or other infective agents.
Greiner Bio-One North America, Inc. Toll Free: 800.884.4703
4238 Capital Drive Tel: 704.261.7800
Monroe, NC 28110 Fax: 704.261.7899
www.gbo.com/bioscience E-Mail: info@us.gbo.com GBOI/NC/3000/V6/05/07