I n a recent study, a method was described for isolating plaque-forming cells (PFC) z in considerable numbers for electron microscope examination (1). In earlier studies involving small numbers of P F C obtained from an active rabbit lymph node (2), and from lymph emerging from such a node (3), all the P F C isolated from the lymph had been found to be in the lymphocytic category. Since this was in contrast to P F C of the lymph node, which included both lymphocytes and plasma cells, a study of PFC of efferent lymph was undertaken, using the method referred to for isolation of PFC in numbers sufficient to justify a picture of the population of cells involved. This question was of particular interest since in our recent study of some hundreds of rosetteforming cells (RFC) and P F C of lymph nodes and spleens, the plasma ceils constituted the great majority of the PFC (1).
Materials and MethodsCollection of Lymph.~4 days after a single injection of 0.5 ml of 50% sheep red blood cells (SRBC) in each hind footpad, rabbits were anesthetized, the skin of the inner aspect of the knee was incised, the semitendinosus and semimembranosus muscles were cut, and each popliteal lymph node was exposed. A ligature was placed, but not tied, under the exposed efferent lymphatic vessel, and the leg was exercised in a prescribed manner. The ligature was then tied and 12 min later, following an exact repetition of the exercise, the lymphatic vessel was entered distal to the ligature with a 27 gauge needle. The lymph was collected in a syringe moistened with heparin solution, 1000 USP units/ml, and centrifuged at 1200 rpm for 5 min. The cell sediment was resuspended in Earle's balanced salt solution containing 5% rabbit serum albumin.Hemolytic Antibody Plaques.--In order to obtain PFC in considerable numbers, a thinlayer agar plating method was used, which was an adaptation of a method recently described (4). Briefly, preliminary plating was done to approximate the number of PFC per milliliter of the suspension, and then a plating with an appropriately higher concentration of lymph