PBMC were cultured as described in Material and Methods to reactivate MBC. The cells were then plated, at several dilutions, onto 96-well nitrocellulose dishes coated with 1µg/well of recombinant influenza HA antigens, KLH (negative control), or antibody against human IgG. After a 4-hour culture, the cells were washed off and the plates developed with a second anti-human IgG antibody conjugated to alkaline phosphatase, and TCIP/BCP substrate. Spots were counted on an ImmunoSpot automated plate reader. Plates were set up such that PBMC from all the time points from a single subject were on the same plate. The wells shown are from a single subject, though representative of all subjects responding in the assay.