A procedure was described for the separation of lymphocytes, polymorphonuclear (PMN) leukocytes and monocytes on Garvin’s glass bead columns. Dextran-sedimented leukocyte suspensions were added to columns and incubated at 37 C. Lymphocytes were eluted with fresh serum. The columns were then washed completely free of erythrocytes, platelets and lymphocytes while PMN leukocytes and monocytes continued to adhere. PMN leukocytes and monocytes were released from the glass with EDTA. Monocytes appeared in the effluents somewhat later than the PMN leukocytes, permitting a separation.

A fresh serum, heat labile, Ca+ +- and Mg+ +-requiring PMN leukocyte and monocyte adherence promoting factor was demonstrated and found to be essential to the procedure.

The viability of column-separated cells was shown by their non-staining with trypan blue, motility, phagocytic ability, oxygen consumption, and survival or development in tissue culture.

In tissue culture, macrophages developed only from monocytes, whereas Nowell’s blast-like, dividing, phytohemagglutinin cells were produced only in cultures containing lymphocytes.

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