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MONOCYTE AND NEUTROPHIL ISOLATION
This procedure describes the method used for isolation of monocytes and neutrophils used in both the chemotaxis assay and microbicidal assay described below.
Heparinized whole blood is layered on top of a density gradient material (Ficoll/Hypaque) and subjected to a centrifugal force. Since erythrocytes (RBC) and PMN are more dense than the
gradient material, they penetrate the gradient material and sediment to the bottom of the centrifuge tube. The lighter mononuclear cells (lymphocytes and monocytes) sediment to the plasma-density gradient interface. At equilibrium, the mononuclear cells are carefully aspirated and saved. The remaining plasma and gradient material are aspirated down to the RBC interface. The mononuclear cells are layered onto gelatin-coated flasks, allowing the monocytes to adhere.
The nonadherent mononuclear cells (lymphocytes) are carefully decanted. The adherent monocytes are dislodged from the gelatin-coated surfaces with vigorous pipeting. The RBC
cushion (which also contains PMN) is resuspended to the initial whole blood volume. Dextran 500 is added to a final concentration of 1% and mixed. The RBC are allowed to sediment to the bottom of the centrifuge tube. The PMN remain in suspension. The PMN-rich supernatant is carefully aspirated.