Lymphocyte Separation Medium (LSM) is a sterile, iso-osmotic polysucrose and diatrizoate solution with low viscosity originally designed for the in vitro isolation of lymphocytes from diluted whole blood. LSM is based on the adapted method of isolating lymphocytes using centrifugation techniques by BÆyum in which diluted defibrinated blood is layered on a solution of Sodium Metrizoate and Dextran or Ficoll® and centrifuged at low speeds for 30 minutes.
Differential migration following centrifugation will result in the formation of several cell layers. Mononuclear cells (lymphocytes and monocytes) and platelets will be contained in the banded plasma-LSM interphase due to their density. The pellet that is formed contains mostly erythrocytes and granulocytes, which have migrated through the gradient to the bottom of the tube. Lymphocytes are recovered by aspirating the plasma layer and then removing the cells. Excess platelets, LSM, and plasma can then be removed by cell washing.
Mediatech's Lymphocyte Separation Medium (Cat.No. 23-25-072-CI or CV) is a sterile filtered solution containing 106.91 g/L of Diatrizoic Acid and 68.181 g/L Polysucrose 400 at a density of 1.077-1.080 g/mL @ 20 C. The solution has an osmolarity of 290 ± 20 mOsm and a pH of 7.0±2.0. Sodium Hydroxide is added as needed to adjust pH.
INSTRUCTIONS FOR USE
Lymphocyte Separation Medium is designed for the simple, rapid isolation of lymphocytes from whole blood that has been diluted and treated with anti-coagulant or defibrinating agent. NOTE:For best results use blood drawn less than 2 hours before. Do not use blood more than 24 hours from when it was drawn.
- 1. Thoroughly mix the LSM by inverting the bottle gently.
- 2. Aseptically transfer 3 mL of LSM to a 15 mL centrifuge tube.
- 3. Mix 2 mL of defibrinated or hepranized blood with 2 mL of physiological saline (Cat.No. 23-21-040-**) or balanced salt solution (Cat.No. 23-21-031-**).
- 4. Carefully layer the diluted blood over 3 mL of LSM (room temperature) in a 15 mL centrifuge, creating a sharp blood-LSM interphase. DO NOT MIX! The quality of the separation is dependent upon a sharp interphase between the lymphocytes and the solution.
- 5. Centrifuge the tube at 400 x g at room temperature for 15 to 30 minutes. Centrifugation should sediment erythrocytes and polynuclear leukocytes and band mononuclear lymphocytes above the LSM as shown in Figure 1.
- 6. Aspirate the top layer of clear plasma to within 2 - 3 mm above the lymphocyte layer.
- 7. Aspirate the lymphocyte layer plus about half of the LSM layer below it and transfer it to a centrifuge tube. Add an equal volume of buffered balanced salt solution to the lymphocyte layer in the centrifuge tube and centrifuge for 10 minutes at room temperature (18 to 25°C) at a speed sufficient to sediment the cells without damage, i.e., 160 - 260 x g. Washing the cells removes LSM and reduces the percentage of platelets.
- 8. Wash the cells again with buffered balanced salt solution (Cat.No. 23-21-031-**) and resuspend in the appropriate medium for your applications.
|23-25-072-CI||Lymphocyte Separation Medium1 |
Density = 1.077-1.080 g/mL at 20°C
|23-25-072-CV||Lymphocyte Separation Medium1 |
Density = 1.077-1.080 g/mL at 20°C
|23-61-196-RM||Polysucrose 400 (generic form of Ficoll® 400)2, Powder||A||A||Res|
|23-61-196-RO||Polysucrose 400 (generic form of Ficoll® 400)2, Powder||A||A||Res|
F = -5 to -20°C
R = 2 to 8°C
A = 15 to 30°C
* = Glass
** = Flexible Media Bag
I = Sterile, IVD Product
NI = Sterile, Research Use Only, Not for IVD Use
IVD = In Vitro Diagnostic
Res = Research Use Only, Not for IVD Use
1 Similar to Ficoll-Paque®, Histopaque®, LymphoSep®, and LSM®
2 Similar to Mono-Poly Resolving Medium®
Histopaque is a registered trademark of Sigma.
LSM, LymphoSep, and MonoPoly Resolving Medium are registered trademarks of ICN Pharmaceuticals