ANDC DU/Biology Protocols/Isolation Mononuclear cells

=Experiment: Isolation of mononuclear cells by Ficoll-Paque PLUS=

Introduction
Ficoll-Paque™ PLUS is a sterile, ready to use density gradient medium for purifying lymphocytes in high yield and purity from small or large volumes of human peripheral blood, using a simple and rapid centrifugation procedure based on the method developed by Bøyum (1). Ficoll-Paque PLUS can also be used to prepare purified lymphocytes from sources other than human peripheral blood. Separation of normal whole human peripheral blood by this procedure typically yields a lymphocyte preparation with: • 60 + 20% recovery of the lymphocytes present in the original blood sample, • 95 + 5% monocular cells, • >90% viability of the separated cells, • 3 + 2% granulocytes, • 5 + 2% erythrocytes and • <0.5% of the total platelet content of the original blood sample.

Ficoll-Paque PLUS

Ficoll-Paque PLUS is an aqueous solution of density 1.077 + 0.001 g/ml containing 5.7 g Ficoll 400 and 9 g sodium diatrizoate with 0.0231 g calcium disodium ethylenediamintetraacetic acid in every 100 ml. Ficoll 400 is a synthetic high molecular weight (Mw 400 000) polymer of sucrose and epichlorohydrin which is readily soluble in water. The molecules of Ficoll 400 are highly branched, approximately spherical and compactly coiled with a Stokes’ radius of a about 10 nm. Ficoll 400 has a low intrinsic viscosity (17 ml/g) compared with linear polysaccharides of the same molecular weight (cf. dextran Mw 400 000: /h/ 49 ml/g) and solutions of Ficoll 400 have low osmotic pressures. Sodium diatrizoate is a convenient compound to use with Ficoll 400 since it forms solutions of low viscosity with high density. Sodium diatrizoate (Mr 635.92) is the sodium salt of 3,5- diacetamido-2,4,6-triiodobenzoic acid. Since sodium diatrizoate is light-sensitive, Ficoll-Paque PLUS must be stored protected from light. The function of sodium diatrizoate in Ficoll-Paque PLUS is to provide the optimal density and osmolarity necessary for the efficient removal of other cells from the lymphocytes. Ficoll-Paque PLUS is supplied as a sterile solution in a bottle with a rubber septum closure. To maintain sterility, aseptic techniques should be used when withdrawing solution and the rubber septum should not be removed. Ficoll-Paque PLUS should be stored between 4 °C and 25 °C and protected from direct light. Storage of unopened bottles in the dark will increase their shelf-life. Deterioration of Ficoll-Paque PLUS is indicated by the appearance of a distinct yellow colour or particulate material in the clear solution. Ficoll-Paque PLUS showing such deterioration should be discarded.

The separation principle
Lymphocyte isolation using Ficoll-Paque PLUS is based on methodology established through the extensive studies of Bøyum (1,2,3) and investigations carried out in different laboratories. Separation media consisting of a mixture of Ficoll 400 and an iodinated density gradient medium such as sodium diatrizoate have been very widely used for purifying human lymphocytes following the publication of Bøyum’s pioneering work in 1968. For lymphocyte separation, defibrinated or anticoagulant-treated blood is diluted with an equal volume of balanced salt solution and layered carefully over Ficoll-Paque PLUS (without intermixing) in a centrifuge tube. After a short centrifugation at room temperature (typically at 400 gav for 30–40 min) lymphocytes, together with monocytes and platelets, are harvested from the interface between the Ficoll-Paque PLUS and sample layers. This material is then centrifuged twice in balanced salt solution to wash the lymphocytes and to remove the platelets. Several factors contribute to the success of this separation. On centrifugation, cells in the blood sample sediment towards the blood/Ficoll-Paque PLUS interface, where they come in contact with the Ficoll 400 present in Ficoll-Paque PLUS. Red blood cells are efficiently aggregated by this agent at room temperature. Aggregation increases the rate of sedimentation of the red cells, which rapidly collect as a pellet at the bottom of the tube, where they are well separated from lymphocytes. Granulocytes also sediment to the bottom of the Ficoll-Paque PLUS layer. This process is facilitated by an increase in their densities caused by contact with the slightly hypertonic Ficoll-Paque PLUS medium. Thus, on completion of centrifugation, both granulocytes and red blood cells are found at the bottom of the tube, beneath the Ficoll-Paque PLUS. Lymphocytes, monocytes, and platelets are not dense enough to penetrate into the Ficoll-Paque PLUS layer. These cells therefore collect as a concentrated band at the interface between the original blood sample and the Ficoll-Paque PLUS. This banding enables the lymphocytes to be recovered with high yield in a small volume with little mixing with the Ficoll-Paque PLUS medium. Washing and centrifugation the harvested cells subsequently removes platelets, any contaminating Ficoll-Paque PLUS and plasma. The resulting cell suspension then contains highly purified, viable lymphocytes and monocytes and is suitable for further studies.

A recommended standard method
Lymphocyte purification using Ficoll-Paque PLUS can be carried out over a wide range of blood sample volumes. With its high yield, this method can be adapted to the processing of very small amounts of blood, such as may be obtained from children. Because of its rapidity and simplicity it is also the method of choice for emergency tissue typing procedures (4). For maximum reproducibility of separation it is recommended that a standardized procedure be used. The following procedure has been evaluated in laboratories and is recommended for separation of normal blood samples on Ficoll-Paque PLUS. Simple changes can easily be made to suit a particular centrifugation system. To standardize the technique, blood volume and diameter of the centrifuge tube should be chosen first. These factors determine the height of the blood sample in the tube and consequently the centrifugation time. Increasing the height of the blood sample in the tube increases red cell contamination. The separation is, however, not appreciably affected by changing the diameter of the tube. Hence a larger volume can be separated with the same degree of purification in a tube of larger diameter if the height of the blood sample in the tube and the separation time are kept constant. The yield and degree of purity of the lymphocytes depend to a considerable extent on the efficiency of red cell removal. When erythrocytes in whole blood are aggregated, some lymphocytes are trapped in the clumps and therefore sediment with the erythrocytes. This tendency to trap lymphocytes is reduced by diluting the blood. Dilution gives a better lymphocyte yield and reduces the size of the red cell clumps. Aggregation of erythrocytes is enhanced at higher temperatures (37°C), which consequently decreases the yield of lymphocytes. At lower temperatures (4°C), however, the rate of aggregation is decreased but the time of separation is increased, which also decreases the yield of lymphocytes. A compromise temperature of 18–20 °C gives optimal results.

Equipment and solutions required:
1. Two 10 ml glass test-tubes for each blood sample to be processed. The test-tubes should be siliconized 2. Balanced salt solution. At least 20 ml for each sample to be processed. The balanced salt solution may be prepared from two stock solutions, A and B.

Solution A Anhydrous D-glucose        5.5 x 10-3 M (0.1%)         1.0 g/l CaCl2.2H2O                       5.0 x 10-3 M                     0.0074 g/l MgCl2.6H20                       9.8 x 10-4 M                     0.1992 g/l KCl                                    5.4 x 10-3 M                     0.4026 g/l TRIS                                  0.145 M                          17.565 g/l Dissolve in approximately 950 ml distilled water and add conc. HCl until pH is 7.6 before adjusting the volume to 1 liltre.

Solution B NaCl                                          0.14 M                     8.19 g/l

To prepare the balanced salt solution, mix 1 volume of solution A with 9 volumes of solution B. Prepare the solution freshly each week. Other standard salt solutions may be used. 3. Pasteur pipettes (3 ml). One for each sample to be processed. These pipettes should be siliconized 4. A low speed centrifuge. 5. Glass centrifuge tubes. Two centrifuge tubes for each blood sample to be processed. Internal diameter approximately 1.3 cm, volume 15 ml. The centrifuge tubes should be siliconizied. 6. Ficoll-Paque PLUS. 3 ml for each sample being processed. 7. Syringe with needle. Needed for withdrawing Ficoll-Paque PLUS from the bottle under aseptic conditions. 8. Trypan blue 9. Microscope 10. Hemacytometer chamber

Preparation of the sample:
Fresh blood should be used to ensure high viability of isolated lymphocytes. Prepare the sample at +18 to +20 °C. 1. To a 10 ml test-tube add 2 ml of defibrinated- or anticoagulant-treated blood and an equal volume of balanced salt solution (final volume 4 ml). 2. Mix by drawing the blood and the buffer in and out of a Pasteur pipette.

Procedure for isolation of lymphocytes: 1. Remove the blue cap on the bottle of Ficoll Paque PLUS. 2. Invert the bottle of Ficoll-Paque PLUS several times to ensure mixing. Using the syringe with needle attached, pierce the septum and withdraw the required volume of Ficoll-Paque PLUS (3 ml for each centrifuge tube) from the inverted bottle. If this method is employed, each bottle will deliver at least 100 ml Ficoll-Paque PLUS. 3. Add Ficoll-Paque PLUS (3 ml) to the centrifuge tube. 4. Carefully layer the diluted blood sample (4 ml) onto the Ficoll-Paque PLUS Important: While layering the sample do not mix the Ficoll-Paque PLUS and the diluted blood sample. 5. Centrifuge at 400gav for 30–40 min at 18–20 °C. 6. Draw off the upper layer using a clean Pasteur pipette, leaving the lymphocyte layer undisturbed at the interface. Care should be taken not to disturb the lymphocyte layer. The upper layer, which contains the plasma, may be saved for later use.

Washing lymphocytes free of platelets: 1. Using a clean Pasteur pipette transfer the lymphocyte layer to a clean centrifuge tube. It is critical to remove all the material at the interface but in a minimum volume. Removing excess Ficoll-Paque PLUS causes granulocyte contamination; removing excess supernatant results in platelet contamination. 2. Add at least 3 volumes (6 ml) of balanced salt solution to the lymphocytes in the test-tube. 3. Suspended the cells by gently drawing them in and out of a Pasteur pipette. 4. Centrifuge at 60–100 gav for 10 min at 18–20 °C. 5. Discard the supernatant. 6. Suspend the lymphocytes in 6–8 ml balanced salt solution by gently drawing them in and out of a Pasteur pipette. 7. Centrifuge at 60–100 gav for 10 min at 18–20 °C. 8. Discard the supernatant. The lymphocytes should now be suspended in the medium appropriate to the application.

Staining and counting of Lymphocytes:
1. Remove 10 :l of well-mixed cell suspension into a well of 96 well plate. Add 10 :l of Trypan blue. Mix well, and load a hemacytometer chamber. 2. Under the microscope count the number of cells in the central 25 squares. Multiply by 2 (dilution factor with trypan blue) and by 104 = cell number/ml.

For example: a) Cells per ml 28 cells x 2 x 104 =256x104 = 2.56x106/ml

b) PBMC total 28 cells x 2 x 104 =256x104 = 2.56x106/ml x final volume = PBMC total

Notes: Preparation of glassware. All glassware that comes in contact with the sample should be siliconized before use. Glassware should be immersed in a 1% silicone solution for 10 seconds (where no specific coating procedure is recommended by the manufacturer), washed thoroughly with distilled water and dried in an oven. The best siliconizing fluids are those based on dimethyldichlorosilane dissolved in an organic solvent. Anticoagulants, Heparin, EDTA, citrate, acid citrate dextrose (ACD), and citrate phosphate dextrose (CPD) may be used as anticoagulants for the blood sample. Bøyum has found that a slightly purer lymphocyte preparation is obtained using EDTA instead of heparin as anticoagulant (3).

References:
1. Isolation of mononuclear cells and granulocytes from human blood. (Paper IV). Bøyum, A. Scand J Clin Lab Invest 21 Suppl, 97, 77–89 (1968). 2. Isolation of leucocytes from human blood - further observations. (Paper II). Bøyum, A. Scand J Clin Lab Invest 21 Suppl, 97, 31–50 (1968). 3. Isolation of lymphocytes, granulocytes and macrophages. Bøyum, A. Scand J Immunol 5 Suppl, 5, 9–15 (1976). 4. Micromethod for rapid separation of lymhocytes from peripheral blood. Fotino, M., Merson, E.J., Allen, F.H. Ann Clin Lab Sci, 1, 131–133 (1971).