ELISpot assay of memory B cell IgG secretion was performed as previously described
25 (link). Immobilon P membrane-based 96-well plates (Millipore, Billerica, MA) were coated overnight at 4°C with 10
µg/mL H3N2 HA in phosphate-buffered saline (PBS) (40
µl/well). PBS only was added to the negative-control wells. Plates were blocked with complete PRMI 1640 medium. Cells were plated at a density of 10
6 per well in U-bottom plates and stimulated with CpG
2006 ODN, six days after stimulation with A/Vic11 (H3N2, IRR catalog No: FR-1041). B cells were resuspended in complete medium containing either alkaline phosphatase-conjugated goat anti-human IgG (H-L) (KPL, Gaithersburg, MD) at 0.2
µg/mL and incubated for 5h at 37°C in 5% CO
2. The plates were washed, and then HA antibody secreting cell spots were developed with an alkaline phosphatase substrate kit (Vector Laboratories, Burlingame, CA). Spots were counted using a CTL ImmunoSpot plate reader and counting software (Cellular Technology Limited, Cleveland, OH).
25 (link). Immobilon P membrane-based 96-well plates (Millipore, Billerica, MA) were coated overnight at 4°C with 10
µg/mL H3N2 HA in phosphate-buffered saline (PBS) (40
µl/well). PBS only was added to the negative-control wells. Plates were blocked with complete PRMI 1640 medium. Cells were plated at a density of 10
6 per well in U-bottom plates and stimulated with CpG
2006 ODN, six days after stimulation with A/Vic11 (H3N2, IRR catalog No: FR-1041). B cells were resuspended in complete medium containing either alkaline phosphatase-conjugated goat anti-human IgG (H-L) (KPL, Gaithersburg, MD) at 0.2
µg/mL and incubated for 5h at 37°C in 5% CO
2. The plates were washed, and then HA antibody secreting cell spots were developed with an alkaline phosphatase substrate kit (Vector Laboratories, Burlingame, CA). Spots were counted using a CTL ImmunoSpot plate reader and counting software (Cellular Technology Limited, Cleveland, OH).