Tech Tips

How to isolate lymphocytes from whole blood

Does your research require you to isolate lymphocytes including T cells, B cells or Natural killer (NK) cells?

If you are performing research or clinical testing that involves studying or characterising the function or numbers of different lymphocytes, then you may be familiar with the process of isolating lymphocytes from whole blood using density gradient centrifugation.

 

Corning® offers a medium that can be used to isolate lymphocytes from diluted whole blood called Lymphocyte Separation Medium (LSM). 

Corning LSM is designed for the simple, rapid isolation of lymphocytes from whole blood that has been diluted and treated with anti-coagulant or defibrinating agent.

 

Principle of the Procedure

LSM is a sterile, iso-osmotic polysucrose and diatrizoate solution with low viscosity.  The procedure is based on the adapted method of isolating lymphocytes using centrifugation techniques by Boyum in which diluted defibrinated blood is layered on a solution of sodium metrizoate and dextran or polysucrose 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.

 

Instructions for Use

Note: For best results use blood drawn less than 2 hours before. Do not use blood that was drawn more than 24 hours prior to use.

 

Step 1: LSM must be at room temperature. Thoroughly mix the LSM by inverting the bottle gently.

Step 2: Aseptically transfer 3 mL of LSM to a 15 mL centrifuge tube.

Step 3: Mix 2 mL of defibrinated or heparinized blood with 2 mL of PBS or balanced salt solution.

Step 4: Carefully layer diluted blood on top of the LSM, creating a sharp blood-LSM interphase. Do not mix. The quality of the separation is dependent upon an interphase between the lymphocytes and the solution.

Step 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.

Step 6: Aspirate the top layer of clear plasma to within 2-3 mm above the lymphocyte layer and discard.

Step 7: Aspirate the lymphocyte layer and dilute with buffered balanced salt solution (~3 volumes) into a new centrifuge tube and centrifuge for 10 minutes at room temperature at a speed sufficient to sediment the cells without damage, e.g., 160-260 x g. Washing the cells removes the LSM and reduces the percentage of platelets.

Step 8: Wash the cells again with buffered balanced salt solution and resuspend in the appropriate medium for your applications.

 

Troubleshooting FAQ

Problem Possible Cause Solution
RBC Contamination Saline diluted blood of low viscosity plasma Increase centrifuge speed
Temperature incorrect Adjust to proper temperature
No defined or distinct mononuclear layer Volume of blood too low Add more blood or dilute blood (1:2) with saline solution
Centrifuge speed too low Increase time or speed of centrifugation

 

Products you need

LSM

SKU Unit Size Product Description
25-072 100 mL, 500 mL Lymphocyte Separation Medium (LSM)

 

Before Isolation:

SKU Unit Size Product Description
46-034-CI 100 mL 0.5M EDTA, pH 8.0
46-013-CM 1 L 10X Phosphate Buffered Saline (PBS), pH 7.4, Liquid
20-021-CV 500 mL Hanks’ Balanced Salt Solution, 10X
20-030-CV 500 mL Dulbecco’s Phosphate-Buffered Saline, 10X

 

After Isolation:

SKU Unit Size Product Description
10-040-CV 500 mL RPMI 1640, w/ L-glutamine
21-031-CV 500 mL Dulbecco’s Phosphate-Buffered Saline, 1X

 

 References

  • Boyum A., Separation of white blood cells. Nature 204, 793-794, (1976).
  • Boyum A., Isolation of mononuclear cells and granulocytes from human blood. Scand.J.Clin.Invest. 21 Suppl. 97:77 (1968).
  • Harris, R. and Ukaylofo E.V., Rapid preparation of lymphocytes for tissue typing Lancet 2. 327, (1969).
  • Thornsby, E. and Bratlie, A., A rapid method for preparation of pure lymphocyte suspensions. In Histocompatibility Testing, P.I. ed. Munksgaard, Copenhagen, p. 664-665, (1970).
  • Ting, A. and Morris, P.J. A technique for lymphocyte preparation from stored heparinized blood. Vox Sang 20. 561, (1971).