A one-step method for In-vitro Isolation of pure lymphocytes.
INTRODUCTION TO THE TECHNIQUE
Early procedures for separating lymphocytes from whole blood involved mixing the blood with a compound, which aggregates the erythrocytes. The clumped aggregates then settle to the bottom, leaving the lymphocytes in the upper layer. Granulocytes are also sedimented since they are larger than lymphocytes, and show a slight tendency to aggregate. Boyum (1964) devised a system in which the aggregating agent is not mixed with the blood. The aggregating agent is mixed with a compound of high density (eq. Sodium Diatrizoate), and the whole brood is carefully layered on top. As erythrocytes reach the interface of the two liquid phases, they contact the aggregating agent, form large clumps, and sediment to the bottom, the granulocytes also sediment out, leaving a high concentration of lymphocytes just above the interface.
Using Ficoll® as the aggregating agent, Boyum (1 968) devised a one-step centrifugal technique for isolation of lymphocytes. Thornsby and Brallie (1970) slightly modified this technique for lymphocyte isolation prior to cyototoxicity tests and lymphocyte culturing. Harris and Ukayiofo (1969), and Ting and Morris (1971) emphasized the reliability of this procedure for fresh blood, stored anti-coagulated blood, and cadaver blood.
Isolymph® is an aqueous solution of Sodium diatrizoate and Ficoll 400®*, having a density of 1-077 ± 0.001 g/ml. (9.0g Sodium diatrizoate and 5.7 g Ficoll 400® per 100 mL). Isolymph® is supplied in a clear, 100 ml bottle with a rubber septum closure for sterile syringe withdrawal. Sodium diatrizoate is the sodium salt of 3, 5.Diacetamido.2.4, 6,-tri-iodobenzoic acid. It forms solutions of low viscosity and high density making it ideal for this application. Since it is light sensitive, Isolymph® must be stored protected from light. Room temperature storage is sufficient, but storage at 4-6°C will increase shelf life.
MATERIALS AND SOLUTIONS
- Two siliconized 10 ml glass tubes for each sample. (See notes).
- Siliconized Pasteur pipettes. (See notes).
- Two siliconized glass centrifuge tubes for each sample (A convenient size tube is 15 ml, internal diameter 1.3 cm) for larger sample volumes see notes.
- A centrifuge.
- A sterile syringe and needle for sterile withdrawal of Isomlymph from its container.
- Isolymph® 3 ml per sample. (For larger samples, see notes).
- 20 ml balanced salt solution for each same. (See notes).
Fresh blood is recommended to ensure high lymphocyte viability.
- Put 2 ml of defribrinated or anticoagulant treated blood into a siliconized 10 ml tube and add an equal amount of balanced salt solution.
- Mix by drawing in and out of a Pasteur pipette.
* Ficoll 400® is a registered trademark of Pharmacia Fine Chemicals AB., Uppsala Sweden,
- Peel back the center tab at the Isolymph® vial cap. Invert the bottle and use a sterile syringe and needle to withdraw Isolymph®.
- Add 3.0 ml Isolymph® to each centrifuge tube,
- Very gently layer the prepared blood sample over the Isolymph®. Do not mix the two layers.
- Centrifuge the sample at 400 x g for 30-40 minutes at 18-20°C,
- Carefully thaw off the upper layer using a Pasteur pipette, leaving the lymphocyte layer intact (This upper layer contains plasma and platelets).
- Using a clean Pasteur pipette, carefully remove the lymphocyte layer. Be certain to remove all the lymphocyte layer but a minimum of the lsolymph layer since this contains granulocytes
WASHING PROCEDURE FOR LYMPHOCYTES
- Transfer the lymphocyte layer to a clean centrifuge tube.
- Add at least three volumes of TRIS-balanced salt solution to the tube1 and suspend the lymphocytes by gently drawing them in and out of a Pasteur pipette,
- Centrifuge at 60-100 x g for 10 minutes at l8-20o C
- Remove the supernatant.
- Add 6-8 ml of TRIS-balanced salt solution and resuspend the cells as above.
- Centrifuge at 60-100 x g for 10 minutes at 18-20oC.
- Remove the supernatant.
- Resuspend the lymphocytes in a suitable medium for your subsequent work.
- All glassware coming into contact with the blood sample should be siliconized. Immerse In a 1% silicon solution for 10 seconds, wash with distilled water (6 times) and oven dry.
- To ensure reproducibility of the lymphocyte technique, the same volume of blood and centrifuge tubes of uniform dimensions must be used. This is critical since the higher the column of blood, the longer the centrifugation time required and the greater the erythrocyte contamination, For larger samples use a tube of larger diameter to keep the height of the blood sample and Isolymph constant.
- A centrifugation temperature of 18-20oC is optimal at higher temperatures (37°C) red cell aggregation is increased, but lymphocyte viability is decreased. At lower temperatures (4°C) the time of separation is increased which decreases lymphocyte viability.
- Defibrinated blood is recommended for this procedure, but anticoagulants such as ACD, CPD, Heparin, EDTA, or Citrate may be used
- Preparation of TRIS-balanced salt solution.
|D-Glucose (anhydrous)||1 .0|
|CaCl2 • 2HO||0.0074|
Working TAIS-balanced salt solution:
Mix 1 volume solution I with 9 volumes of solution II. Prepare fresh weekly.
6. The erythrocyte contamination in the lymphocyte suspension using this procedure is usually 1-5% of the total cell number
- Boyium, A (1964): Separation of White Blood Cells. Nature 204, p. 793.
- Boyium. A. (1968): Separation of Leucocytes from Blood end Bone Marrow. Scand. J.Clin. Lab Invest. 21 Suppl. 97
- Favour C B. (1964): Antigen-antibody Reaction in Tissue Culture. Immunological Methods, Ed. J. R. Ackroyd, p.195-223. Blackwell Scientific Publ., Oxford.
- Harris, R. and Ukaejiofo. E. V. (1969): Rapid Preparation for Lymphocytes for Tissue Typing. Lancet 327. 7615.
- Ting. A and Morris. P. J (1971): A Technique for Lymphocyte Preparation from stored Heparinized Blood. Vox Sang, 20. 561.
- Thorsby E. and Bratlie, A (1970): A Rapid Method for Preparation of Pure Lymphocytes Suspensions. Histocompatibility testing. 1970 Ed P. I. Terasaki. p. 655 Munksgaard Copenhagen