Isolation of Human Polymorphonuclear Leukocytes (Granulocytes) from a Leukocyte-Rich Fraction

Human peripheral blood polymorphonuclear leukocytes (PMNs) or granulocytes from a leukocyte-rich plasma (LRP) are banded at an interface between two layers of iodixanol. If the denser layer of iodixanol is omitted the PMNs may alternatively be pelleted. The procedure can be adapted to blood from other species by small changes to the density of the two iodixanol layers. The method works optimally with EDTA- or citrate-anticoagulated blood.


INTRODUCTION
The polymorphonuclear leukocyte (PMN) fraction from human peripheral blood comprises principally neutrophils with relatively small numbers of basophils and eosinophils. They have densities predominantly above 1.077 g/ml, with the exception of part of the basophil population, while mononuclear cells (lymphocytes + monocytes) have densities below this value (see Fig. 1). Since the density of erythrocytes significantly overlaps that of neutrophils, it is only possible to use a simple OptiPrep discontinuous gradient to resolve the mononuclear cells and PMNs from Layering LRP over a barrier whose density is 1.077 g/ml, will allow the PMNs to pellet while the mononuclear cells are retained at the interface. However, to avoid pelleting the PMNs, which could lead to aggregation, a dense cushion (1.090-1.095 g/ml) may be placed beneath the 1.077 g/ml layer in order to band the PMNs.
Subsequent to purification, erythrocyte contamination of the PMN fraction can be eliminated by selective osmotic shock either in cold water or in isotonic NH4Cl (see below).

MATERIALS AND EQUIPMENT
OptiPrep (shake gently before use) Diluent: 0.85% (w/v) NaCl, 1 mM EDTA, 20 mM HEPES-NaOH, pH 7.4 Dextran: 6% (w/v) dextran (M r = 400-500 ×10 3 ) in 0.9% (w/v) NaCl (optional) Lysis Buffer (LB): 0.83% (w/v) NH 4 Cl, 10 mM HEPES-NaOH, pH 7.0 (optional). 1.8 (w/v) NaCl, 20 mM HEPES-NaOH, pH 7.4 (optional) Plastic conical centrifuge tubes (12-15 ml) Syringe with metal cannula for underlayering Plastic Pasteur pipettes Low-speed (temperature-controlled) centrifuge with swinging-bucket rotor 4. Underlayer the 5 ml of LRP with 3-4 ml of 1.077 g/ml solution and the same volume of EITHER 1.090 g/ml OR 1.095 g/ml. 5. Centrifuge at 18-22 o C for 25 min at 800g. 6. Using the Pasteur pipette, harvest the two bands in turn; the mononuclear cells from the upper interface and then the PMNs from the lower interface (see Fig. 2). 7. Dilute the PMN suspension with an equal volume of Diluent and collect the cells by centrifugation at 250g for 10 min. 8. Resuspend the pellet in a suitable medium (see Note 4). 9. To remove erythrocyte contamination from the PMNs, resuspend the cell pellet in 3 ml of LB and incubate at 37 o C for 7 min OR resuspend the PMNs in 3 ml ice-cold distilled water, then after 30 s add an equal volume of the 1.8% NaCl solution (see Note 4). 10. Harvest by centrifugation and resuspend in a suitable medium.

NOTES
1. The blood should be processed within 6 h of drawing. Ideally the erythrocytes should be removed as soon as possible after drawing but the subsequent gradient separation of the mononuclear cells and PMNs can be carried out up to 6 h later. 2. If the density of this cushion is 1.090 g/ml, a small percentage of the neutrophils and most of the eosinophils will sediment through this layer. If a density of 1.095 g/ml is chosen, virtually all of the PMNs will be retained by the high-density barrier. On the other hand, fewer of the residual erythrocytes in the LRP will contaminate the PMN band using the lower density cushion. 3. For nonhuman blood, it may be necessary to adjust the density of one or both of the layers.
For rat blood for example, increasing the density of the middle layer to 1.080 g/ml may be of benefit (equivalent to approx. 14.2% (w/v) iodixanol). See Ref. [1] for more information on preparing gradient solutions. 4. The cells should be thoroughly, BUT GENTLY, resuspended in the liquid. The RCF used to sediment the cells in step 7 is deliberately chosen to avoid excessive aggregation in the pellet. A satisfactory suspension of this pellet should be achievable with slow aspiration into and expulsion from a Pasteur pipette or gentle agitation.