Depletion of monocytes from human peripheral blood mononuclear leukocytes: Comparison of the sephadex G-10 column method with other commonly used techniques

Abstract

Several techniques in current use for depletion of human monocytes were critically evaluated in terms of cell yield, efficiency of monocyte depletion, alterations in the proportion of lymphocytes subpopulations and functional activity of the recovered cell in microculture lympocyte proliferation assays. Human peripheral blood mononuclear leukocytes (PBML) isolated by Ficoll-Hypaque (FH) gradients were depleted of adherent monocytes by passage over nylon wool, rayon wool or Sephadex G-10 comlumns. Phagocytic cells were removed by the addition of carbonyl iron to either whole blood or isolated PBML followed by Ficoll-Hypaque gradient centrifugation or application of a magnetic field. It was found that carbonyl iron treatment of whole blood followed by density gradient sedimentation of the ironm containing cells resulted in the best recovery of cells but gradient failed to efficiently deplete esterase-positive cells (Mθ). Both nylon wool and rayon wool columns removed esterase-positive cells to a relatively low level; (0.2 and 0.3.%, respectively), although both consistently gave the poorest cell yields of the techniques evaluated. Addition of carbonyl iron to PBML folllowed by removal of cells phagocytizing iron by magnetic field was found to give poor cell yield and poor depletion of Mθ. In contrast, Sephadex G-10 columnsm consistently depleted Mθ to low levels and provided cell yields to nylon and rayon wool columns. In contrast to nylonm wool columns, using Sephadex G-10 columns it was possible to deplete Mθ without altering the proportions of T- and B-lympocytes. Further, a cell population enriched for Mθ could be recovered from the Sephadex G-10 beads using a 0.5% lidocainem solution. It was found that none of the column techniques impaired the ability of the recovered lympocytes to proliferate to the T-cell mitogen phytoemaggluitinin, although responses to recall antigens were eliminated by these techniques, indicating an Mθ requirement for the latter responses. Since the Sephadex G-10 column procedure appeared superior to the other Mθ depletion procedures, further studies were performed to define some of the variables affecting the optimization of this method. Sephadez G-10 columns of 5–6 ml bed volume loaded with 3–5 × 10 7 cells proved optimum for maximum recovery of lymphocytes and depletion of Mθ.