Studies on the production of complement.

The production of complement was investigated using a modification of Jerne's haemolytic plaque technique. Guinea-pig spleen and bone marrow cells were suspended in layers of agarose containing, as indicator cells, sensitized sheep erythrocytes (EA) or intermediate products consisting of EA and some of the components of complement. R reagents or serum, in which certain components of the C′ complex had been inactivated, were added after incubation. No plaque formation due to release of complement components was observed. Bone marrow and spleen cells produced plaques when incubated with sensitized or non-sensitized erythrocytes and a reagent containing cobra venom. A method was developed for the preparation of thin films of agarose in which tissue cells and erythrocytes were suspended in a single cell layer. Single cells and clumps of cells present in the peritoneal exudate of normal guinea-pigs produced plaques when incubated with EA or EAC′ and R. Occasionally, similar clumps of cells also produced plaques with EA or EAC′ and R. The plaque-forming cells appeared to be macrophages. Plaques were not detected with R or R. No plaques were produced by spleen cells under similar experimental conditions. Bone marrow cells produced plaques with both sensitized and non-sensitized erythrocytes after prolonged incubation. Optimal plaque formation occurred when R or R was added before incubation. Plaques were not produced if the tissue cells were pre-incubated in suspension cultures for 2 hours or longer before plating. Treatment of the peritoneal exudate cells with KCN or with dinitrophenol prevented plaque formation.