Isolation of monocytes in human peripheral blood Part 1 by discontinuous density gradient

The isolation of monocytes in human peripheral blood by discontinuous density gradients was studied. Discontinuous density gradients were obtained by successive layering of bovine serum albumins into three layers in 10ml glass tubes. Buffy coat cells were layered on top of a gradient and centrifuged under various conditions of centrifugation. Optimal isolation was achieved by centrifugation on a three layer of 35-32-28% bovine serum albumin at 490g for 60 minutes. The purity, yield and recovery of monocytes at the interface between 35% and 32% bovine serum albumin were 54.0%, 7.0×10(5) and 22.0% respectively. After centrifugation on Conray-Ficoll mixtures, the interface fraction containing lymphocytes was removed and the bottom fraction containing monocytes and other cells was centrifuged under various condition. The optimal condition was on a three layer of 35-32-28% bovine serum albumin at 400g for 40 minutes. The purity of monocytes by this method increased to 75% but the yield and recovery declined to 2.5×10(5) and 4.5% respectively. From these data I concluded that isolation of monocytes from blood only on density gradients was difficult because of a partial overlap between monocytes and lymphocytes in the density distribution profiles