Preparation of lymphocytes from blood

6.2 Preparation of lymphocytes from blood

Differential centrifugation on a density gradient gives high-purity lymphocyte preparations. All equipment and solutions for these techniques must be sterile.

6.2PREPARATION OF LYMPHOCYTES FROM BLOOD

6.2.1 Mouse lymphocytes

Blood is layered onto a density gradient formulated such that only the erythrocytes form a pellet. MATERIALS

Triosil 75 (available commercially, and is sterile) Ficoll Mouse blood sample Tissue culture medium (see Appendix B.6) Centrifuge tubes to prepare gradient Pasteur pipettes

METHOD

1 Prepare a 9.2% w/v Ficoll solution in distilled water and autoclave.

2 Mix 43.4 ml of Ficoll solution and 6.6 ml of Triosil 75 under sterile conditions.

3 Dilute the blood 1 : 1 with sterile tissue culture medium without serum.

4 Layer 5 ml of diluted blood onto 2 ml of the gradient mixture and centrifuge at 300 g for 15 min at 4°C. The lymphocytes should not enter the gradient, but remain at the plasma–density gradient interface as a band.

5 Remove and discard the medium above the lymphocyte band.

6 Recover the lymphocyte band and wash three times by centrifugation (150 g for 10 min at 4°C).

TECHNICAL NOTES • This technique works with heparinized blood. • Discarding the supernatant above the lymphocyte band removes much of the platelet con-

tamination. Washing in tissue culture medium will reduce the numbers of remaining platelets. The supernatant of the first wash will appear turbid because of unsedimented platelets; con- sequently, it is good practice to check by eye that a well-defined pellet has formed before discarding the wash supernatant.

• Do not collect too much of the density gradient; this contains neutrophils.

• Lymphocyte yield is 1–2 × 10 6 /ml of venous blood.

• Commercial preparations of the density gradient are prepared gravimetrically and give more reproducible results. There are many suppliers, e.g. Lymphoprep TM .

6.2.2 Human lymphocytes Preparation of human peripheral blood mononuclear cells from whole blood

In this technique peripheral blood mononuclear cells (PBMC) are isolated from defibrinated or anticoagulated human blood using a density-interface centrifugation technique. It is adapted from Mutch and Westwood (2001).

MATERIALS AND EQUIPMENT Human blood Tissue culture medium, e.g. RPMI 1640 medium supplemented with 2.0 g/l sodium bicarbonate,

2m ML -glutamine, 5 m M 4-(2-hydroxyethyl)piperazine-1-ethane sulphonic acid (HEPES)

C H A P T E R 6: Isolation of cells C H A P T E R 6: Isolation of cells

Centrifuge tubes (50 ml with screw caps) Ficoll–Paque (Ficoll–sodium diatrizoate, r = 1.077; Amersham Pharmacia Biotech) Disposable syringes Flexible 30-cm plastic cannula Pasteur pipette

Note: All equipment and solutions for this technique must be sterile.

METHOD

1 Dilute 20 ml of human defibrinated blood with 10 ml of tissue culture medium, then add to

a 50-ml centrifuge tube.

2 Carefully underlay 13–15 ml Ficoll–Paque using a sterile flexible cannula and disposable syringe, then centrifuge at 450 g for 25 min at 20°C.

3 Remove all but 3 ml of the serum above the layer of PBMC; this can be done carefully using

a sterile pipette.

4 Heat inactivate the serum removed in step 3 by heating for 30 min at 56°C and keep serum for use in step 8.

5 Carefully remove the PBMC layer with minimal underlying Ficoll–Paque and add to a sterile 50-ml centrifuge tube. Samples from a single donor may be pooled (up to 25 ml per 50-ml centrifuge tube).

6 Wash the PBMC by adding an equal volume of tissue culture medium, then gently mix by resuspending the cells.

7 Centrifuge the cells at 150 g for 15 min at 20°C, remove the supernatant and then resuspend the cells in culture medium. (Note that the PBMC layers contain enough serum within the suspension so serum need not be added at this stage.)

8 Resuspend the final pellet in complete medium containing 10% (v/v) heat-inactivated autologous serum (as prepared in step 4).

TECHNICAL NOTE Use autologous serum rather than human AB serum as a supplement for the tissue culture medium for cells to be isolated for proliferative assays, as it avoids the need to pretest the serum. Do not use fetal bovine serum as it is likely to totally destroy the assay!

Human lymphocytes and peripheral blood mononuclear cells

MATERIALS Human blood Endotoxin-free heparin Hank’s balanced salt solution (Sigma) Lymphoprep TM (Nycomed Amersham) Pasteur pipettes 20-ml centrifuge tubes

6.2PREPARATION OF LYMPHOCYTES FROM BLOOD

RPMI 1640 containing 2 m ML -glutamine, 1 m M sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.5 µg/ml fungizone and 10% heat-inactivated pooled human serum (e.g. human AB serum or autologous serum asee Appendix B.6)

METHOD

1 Collect human peripheral blood into tube containing endotoxin-free heparin (10 U/ml blood).

2 Add an equal volume of Hank’s balanced salt solution and mix gently by inversion.

3 Overlay 10 ml aliquots of the diluted heparinized blood onto 5 ml of Lymphoprep TM . Then centrifuge at 800 g for 20 min at 20°C.

4 The middle interface contains the lymphocytes (see Fig. 6.1). Remove cells using a sterile Pasteur pipette, taking care not to disturb the upper layer.

6 Wash the cells with 10 ml of Hank’s balanced salt solution. Pellet the cells by centrifugation at 250 g for 15 min at 20°C.

7 Resuspend the cells for culturing in RPMI 1640 containing 2 m ML -glutamine, 1 m M sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.5 µg/ml fungizone and 10% heat-inactivated serum.

6.2.3 Chicken lymphocytes

Although dextran sedimentation techniques are available to prepare chicken leucocytes, they give poor cell yields. Chicken erythrocytes are much larger than the white cells and so separation by differential centrifugation is possible without a density gradient. Chicken PBMC tend to clump readily under normal conditions and so these are also removed from the plasma.

MATERIAL Chicken blood containing heparin

Fig. 6.1 Lymphoprep: before and

centrifugation

centrifugation

after centrifugation. Lymphocytes are found at the interface.

C H A P T E R 6: Isolation of cells

METHOD

1 Cool the blood to 4°C.

2 Centrifuge at 150 g for 3 min at 4°C.

3 Reduce the centrifugal force to 35 g, and continue spinning for a further 10 min at 4°C.

The supernatant plasma will contain lymphocytes virtually free of all other cells; lymphocyte yield 3–5 × 10 6 /ml whole blood. It is essential to use blood containing heparin, as citrated saline reduces the viscosity of the plasma and reduces cell yield and purity. As cockerel blood has a lower viscosity than hen blood due to its lower lipid content, a loose buffy coat often forms during centrifugation. This should be resuspended by gentle stirring.