Splenocyte t and b cell elispot reagents

Splenocyte T and B cell ELISPOT reagents were obtained from Mabtech (Mariemont, OH). H1HA specific T cells were quantified per manufactures instructions. Positive controls were stimulated with 20 ng/ml PMA (Sigma-Aldrich, St. Louis, MO) and 500 ng/ml ionomycin (Sigma-Aldrich). The H1HA relevant peptide used was a CD8⁺ cell-restricted HA peptide (IYSTVASSL). H1HA-specific antibody secreting cells were detected by coating of MAIPSWU (Millipore, Billerica, MA, USA) plates with the transiently expressed H1HA antigen utilized for ELISA (~1.0 μg/well). Positive spots were visualized on a CTL Imager and counting was performed with Immunospot software (Cellular Technology Ltd., Shaker Heights, OH)

Splenocyte T and B cell ELISPOT reagents were obtained from Mabtech. H1HA specific T cells were quantified per manufactures instructions. Positive controls were stimulated with 20 ng/ml PMA (Sigma-Aldrich) and 500 ng/ml ionomycin (Sigma-Aldrich). The H1HA relevant peptide used was a CD8⁺ T cell epitope (IYSTVASSL). H1HA-specific antibody secreting cells were detected by coating of MAIPSWU (Millipore) plates with the standard H1HA antigen utilized for ELISA (1.0 μg/mL). Positive spots were visualized on a CTL Imager and counting was performed with Immunospot software (Cellular Technology Ltd.)