Isolation of human blood monocytes with Nycodenz, a new non-ionic iodinated gradient medium.

Abstract

Monocytes were separated from human blood with Nycodenz, an iodinated gradient medium. Monocytes have a lower average density than lymphocytes, but because of overlapping efficient separation cannot be achieved on the basis of density differences alone. Thus the isolation procedure was based on the assumption that the low-density fraction of lymphocytes increases its density more than monocytes by expelling water when exposed to an increased osmolarity. Thereby they might pass through a density barrier present initially, whereas the monocytes remain at the top of the gradient layer. Separation fluids with densities ranging from 1.061 to 1.096 g/ml were prepared by mixing Nycodenz with NaCl solutions of various concentrations. EDTA-blood (3 ml) or a leucocyte suspension (2-6 ml) obtained by dextran sedimentation was loaded on 3 ml of separation fluid and centrifuged for 15 min at 1900 rpm. Then the cells in the interface region were collected. At each density level it was possible to obtain an almost pure monocyte suspension (95-98%) by increasing the osmolarity. However, the higher the purity, the lower the monocyte yield. Apparently, the viability of monocytes was not affected, even when subjected to an osmolarity of 600 mosmol. For routine use, it appears that separation fluids with densities from 1.061 to 1.078 g/ml and corresponding osmolarity in the 300 to 410 mosmol range are suitable.