<p>PBMCs from different anonymous blood donors were pre-incubated with vaccine mono-bulk subunits from the B/Brisbane/60/2008 (B/HA pretreatment), or the H3N2 A/Panama/2007/1999 strain (A/HA pretreatment) and then stained HSA, rH3 (from A/Brisbane/10/2007), rH1 (from A/California/07/2009), or B/HA (from B/Brisbane/60/2008), as indicated. <b>A.</b> Staining pattern observed on CD20<sup>+</sup> cells in PBMCs stained with the different rHA bait. The rectangular gates identify brilliant HA+ B-cells; the dotted vertical lines mark the gates used to sort HA<sup>+</sup> B-cells for the ELISPOT assays. <b>B.</b> Expression of the CD27 memory marker on HA<sup>+</sup> and HA<sup>neg</sup> B cells identified based on the sorting gates. <b>C.</b> H3<sup>+</sup> (n = 15,234), H1<sup>+</sup> (n = 6482) and B/HA<sup>+</sup> (n = 26,803) B-cells identified in A were sorted, mixed with autologous CD20<sup>neg</sup> cells (in the ratio of 1∶20, 1∶100 and 1∶33) and activated with CpG and IL-2 for 5 days <i>in vitro</i>. Unsorted PBMCs and CD20<sup>neg</sup> cells mixed with HA<sup>neg</sup> B cells were also cultured in the same manner, as controls. After 5 days cultured cells were harvested and assayed by ELISPOT for the number of cells secreting IgG and IgG specific for mono-bulk subunits from the vaccine strain homologous to the sorting bait. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070620#s2" target="_blank">Results</a> are expressed as numbers of antibody secreting cells (ASC) normalized to 10<sup>6</sup> cultured cells assayed by ELISPOT. Nd indicates undetectable ASC.</p
