Plaque Forming Cell Assay

To analyze IgM-responses, a modified version of the Jerne haemolytic plaque assay (24) was used and details have been described previously (25). Briefly, spleens were removed 5 days after immunization and appropriately diluted single cell suspensions were mixed with agarose (at 45°C), SRBC, and complement (guinea pig serum). The mix was immediately spread on a microscope slide and incubated for 3 h at 37°C in a humid chamber. Haemolytic plaques, each representing a single spleen cell producing IgM anti-SRBC, were counted “blindly.” All dilutions were made in Hank's balanced salt solution, HBSS. Duplicate samples were analyzed. In the event of no detectable plaque forming cells (PFC) in 1:100 of a spleen, the spleen was assigned 50 PFC in order to allow further calculations.