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

A three step method for the purification of normal human blood monocytes is described. The procedure consists of a combination of dextran sedimentation, Ficoll-Isopaque (F-I) centrifugation and isopycnic centrifugation on discontinuous gradients of Percoll. No selective loss of monocytes was observed after the first 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 gradients of Percoll. The best purification of monocytes 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. 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 volumes (500 ml) of blood.