Three-step isolation of human blood monocytes using discontinuous density gradients of percoll

Abstract

A 3-step method for normal human blood monocyte purification is described. The procedure consists of a combination of dextran sedimentation, Ficoll-Isopaque (F-I) centrifugation and isopycnic centrifugation on discontinuous Percoll gradients. No selective monocyte loss was observed after the 1st step and after F-I centrifugation mononuclear cells (MNC) were obtained, of which 20 .+-. 6% were monocytes. The MNC were further separated on hyper-osmotic and iso-osmotic discontinuous density Percoll gradients. The best monocyte purification occurred on hyper-osmotic density gradients and the density interface between 1.074 and 1.066 g/ml yielded 85 .+-. 7% monocytes, 13 .+-. 7% lymphocytes and 1 .+-. 1% granulocytes. Some 77 .+-. 16% of the monocytes obtained after F-I centrifugation, were recovered in this interface. The purified monocytes were viable and retained their capacity to mature into macrophages. The whole procedure takes about 5 h, is reproducible and can be applied to small and large blood volume (500 ml).