Isolation of a monocyte-rich fraction from whole human blood by iodixanol barrier flotation

However because of the requirement to prepare a buffy-coat fraction from whole blood, the yield of monocytes is always compromised by the inevitable loss of leukocytes that occurs during the preparation of such a fraction. This step also adds to the overall time of preparation. Moreover the platelets in the blood also sediment into the buffy coat fraction and the close juxtaposition of platelets and monocytes may lead to activation of the latter. 

The method originally devised by Graziani-Bowering et al [1] has therefore been modified in an attempt to reduce any activation of the monocytes (by platelets or by the isolation procedure itself) to a minimum.

 The method described in this Application Sheet relies on the same principle of separation as that described in Application Sheet C09 (i.e. the more rapid rate of flotation of monocytes compared to lymphocytes) and in both cases the vast majority of the platelets remains in the high-density sample zone and do not co-band with the monocytes. 

The method described in this Optiprep™ Application Sheet is carried out at 4°C; this is tends to reduce activation and minimizes the vesiculation within the cytoplasm of the monocytes that is sometimes observed when the separations are carried out at room temperature. When this vesiculation occurs yields are very low.