USER MANUAL

Cat. No. 091692249, 091692254
Revision Date: 2021-02

LymphoSep™
Lymphocyte Separation MediaINTENDED USE
For in vitro isolation of mononuclearcells (lymphocytes) from peripheral
blood and other blood sources.

SUMMARY OF THE
METHOD
Early methods for isolating lymphocytes
involved mixing blood with a compound
which aggregated the erythrocytes, but
only slightly affected lymphocytes. With
centrifugation, erythrocytes pelleted
due to their increased density andleukocytes were collected from theupper portion of the tube.1
Boyum2 introduced a more convenient
and rapid separation using
centrifugation through a Ficoll®-sodium
metrizoate solution. Diluted blood waslayered over the Ficoll®-sodium
metrizoate solution and centrifuged at
800 x g for 30 minutes. Erythrocytes
and granulocytes sedimented to thebottom of the tube, and lymphocytes
and platelets were collected from theinterface between the two phases.
Modifications to the Boyum formulation
have been made by numerous
researchers.3-6 LymphoSep™,
Lymphocyte Separation Medium (LSM),
manufactured by MP Bio, has a uniqueformulation using the successfulsubstitution of sodium diatrizoate forsodium metrizoate.

PRINCIPLE OF THE
PROCEDURE
Defibrinated or heparinized blood isdiluted with a balanced salt solution,such as PBS or HBSS at a 1:1 ratio,
layered over the separation medium andcentrifuged at 800 x g for 30 minutes.
During centrifugation, differential
migration results in the formation ofseveral cell layers.
A pellet is formed that is comprised
mainly of erythrocytes and granulocytes
which have migrated through thegradient. Due to their density,
lymphocytes and other mononuclear
cells (platelets and monocytes) arefound at the plasma-LSM interface.
Lymphocytes are recovered byaspirating the layer. Further washingremoves platelets, LSM and plasma.

STORAGE Store at room temperature(18-25 °C) in the originalcontainer.

 Stable until the expiration datelisted on the vial.

 Protect from light.
RECOMMENDED
PRODUCTS RPMI 1640 (1X) withL-Glutamine (Cat. No. 0912603)

REAGENTS
LymphoSep is a sterile, filtered solution
containing Polysucrose 400 andsodium diatrizoate at a density of
1.0770-1.0800 g/mL at 20 °C.

 RPMI 1640 with L-glutamine(powder) (Cat. No. 0910601)

 Hank’s Balanced Salts(Cat. No. 091810554)

 DPBS (1X, Cat. No. 091860454)

CAUTION• The material is intended for laboratory use only for the in vitro
separation of lymphocytes from peripheral blood.
• LymphoSep is not intended for in vivo use.
• Do not use if the material is cloudy, has a distinct yellow coloror shows any signs of contamination.

Learn more at www.mpbio.com

USER MANUAL: LymphoSep™ Lymphocyte Separation MediaINSTRUCTIONS FOR USE

7The following is one of many variants ofthe procedure originally described byBoyum2. This procedure was developedfor use with defibrinated or anticoagulant treated blood. For optimalresults, alterations may be necessary
depending on the species the blood ortissue was collected from.
1

Thoroughly mix the LymphoSep
by inverting the bottle gently.

2

Aseptically transfer 3 mLof LymphoSep to a 15 mL
centrifuge tube.

3456Dilute 2 mL of defibrinated or
heparinized blood with 2 mLof DPBS with no Mg2+ or Ca2+.
0.9% NaCl, HBSS or cell culture
media can also be used as adiluent.
Carefully layer the diluted bloodover 3 mL of LymphoSep (roomtemperature) in a 15 mL centrifuge
tube, creating a sharp blood-LSMinterface. Do not mix diluted
blood with the LymphoSep.

8RESULTS

Bovine lymphocytes (with erythrocytesremoved) were obtained using MP Biomedicals’
LymphoSep reagent according to themanufacturer’s protocol. Live/Dead analysis
of isolated cells was performed using theLIVE/DEAD Viability/Cytotoxicity assay kit
from Molecular Probes. 95% of lymphocytes
recovered from 2 mL of whole heparinizedbovine blood were LIVE. Fluorescent
microscopy of cells shows LIVE cells ingreen (40x magnification).

Wash the cells again with DPBSand resuspend in the appropriate
medium for your applications.

NOTE Erythrocytes may be present in
the monoclear fraction at 1-5% of total
cells counted. They can be removed using
a red blood cell lysis solution if necessary.

REFERENCES
Boyum, A. Separation of white blood cells.
Nature. 1964, 204, 793-794.

1 

Boyum, A. Isolation of mononuclear cells andgranulo­cytes from human blood. Scand. J. Clin.
Lab. Invest. Suppl. 1968, 97, 77.

2 PlasmaCentrifuge the tube at 800 x g atroom temperature for 30 minuteswith the brake off. Centrifugation
should sediment erythrocytes
and polynuclear leukocytes andband lymphocytes above theLymphoSep.
Aspirate the top layer of clear
plasma to within 2-3 mm above
the lymphocyte layer.

Aspirate the lymphocyte layer plus
about half of the LymphoSep layer
below it and transfer it to a newcentrifuge tube. Add an equalvolume of DPBS to themononuclear cell layer in thecentrifuge tube and centrifuge for10 minutes at room temperature
at 200 x g (a centrifugation speed
ranging from 160-260 x g can beused–choose a speed sufficient tosediment the cells withoutdamage). Note that washingremoves LymphoSep and reduces
the percentage of platelets.

Harris, R.; Ukaejiofo, E. V. Rapid preparationof lymphocytes for tissue typing. Lancet. 1969,
2, 327.

3 Lymphocyte layer

Thorsby, E.; Bratlie, A. A rapid method for
prepara­tion of pure lymphocyte suspensions.
In Histocompatibil­ity Testing. P.I. Terasaki, ed.
Munksgaard, Copenhagen. 1970, 664-665.

4 

LymphoSep

Ting, A.; Morris, P.J. A technique for lymphocyte
preparation from stored heparinized blood. VoxSang. 1971, 20, 561.

5 RBCsWang, W.; et al. Zika virus infection induces hostinflammatory responses by facilitating NLRP3
inflammasome assembly and interleukin-1βsecretion. Nat. Commun. 2018, 9, 106.

6 

Lymphocyte separation schematicusing LymphoSep

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EUROPE: 00800.7777.9999 | custserv.eur@mpbio.com
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Learn more at www.mpbio.com