The Separation of Human Monocytes from Blood Including Biochemical Observations 1 2

Abstract

The importance of the monocyte and its derivative, the macrophage, is becoming increasingly apparent in many areas of biologic research. In addition to its well-known role in phagocytosis, it likely plays a part in processing antigen in cellular immunity (1) and facilitating the response of lymphocytes to antigen (2), pokeweed mitogen (3), and phytohemagglutinin (4). It is a rich source of colony-stimulating factor for the in vitro growth of colonies of granulocytes and mononuclear cells from human and murine marrow (5). This cell may of use in the immunotherapy of human neoplastic processes. The monocyte is produced in the bone marrow (6), but its precursor cell is yet to be identified. Measurement of the physiological, immunological, and biochemical activities of the normal human monocyte has been hampered by the difficulty of obtaining pure samples in sufficient quantity. Most methods have resulted in monocyte monolayers adherent to a surface where they cannot be accurately counted or easily handled for study. The present study reports the separation of monocytes in high purity and large quantities from normal human blood. It results in cells that are in suspension and, therefore, can be easily quantitated. They can be easily transferred for radioisotopic, morphologic, and immunologic investigations.

Materials and Methods. Two hundred and fifty milliliters of venous blood from normal volunteers was anticoagulated with heparin (12 U/ml of blood) and the erythrocytes sedimented with 5% dextran (Sigma Chem. Co., St. Louis, MO) for 40 min at 37°. The cells in the supernatant were harvested by centrifugation at 190g for 8 min and washed in sterile phosphate-buffered saline (PBS, pH 7.25). The mononuclear cells were initially separated by the method of Bennett and Cohn (7). The cells were suspended in 27% bovine albumin (Cohn fraction V) (Sigma Chem. Co.) in PBS at a cell concentration of 75 × 10⁶ leukocytes/ml.