Surface markers and biological functions of PMNC fractionated by countercurrent centrifugal elutriation

Peripheral blood mononuclear cells (PMNC) were separated by countercurrent centrifugal elutriation (CCE) in PBS-EDTA medium into 13 fractions. Properties associated with B cells were restricted mainly to the first fractions (F2 and F4) while those associated with T cells were confined to fractions F4, F6 and F8. The final fraction (FF) contained more than 90% monocytes while fraction F10 contained 40% monocytes and 60% null cells, and F12 contained 50% monocytes and 50% null cells. Natural killer (NK) cell activity against K-562, a human erythroleukaemia cell line, and against an adherent undifferentiated sarcoma target, was distributed between fractions F6 and F8. Using electron and scanning microscopy and a new single cell liquid cytotoxic assay, we studied membrane interaction and binding of the CCE fractionated cells with the 2 targets. Effector/target cell conjugates revealed interdigitations in the area of cell contact but no membrane fusion, although certain effector cells had a distinct large granulocytic lymphocyte morphology. Furthermore, different cell types from different fractions were able to bind to the targets although the killing was associated with certain fractions only. When purified monocytes from fraction FF were added to F6 and F8, NK activity against both targets was depressed. This inhibition was not reversed by indomethacin, and binding of the targets to the effector cells was unaffected. CCE is a powerful technique which allows the fractionation of PMNC without altering their biological functions, contrary to what is seen with nylon wool or dextran fractionation. In this report, the activity associated with CCE fractions is studied and discussed.