Development of tetramer-associated magnetic enrichment (TAME). (A) PBMCs from healthy donors were stained with HLA-A2.1 tetramers labeled in PE (pre-enriched fraction). Samples were further incubated with anti-PE microbeads, and passed twice over an MS magnetic column. The elution fraction corresponds to the depleted fraction, and retained fraction to the enriched sample. Pre-enriched, enriched, and depleted fractions were then labeled with a flow cytometric panel, and acquisition was performed with a polychromatic flow cytometer. (B) To optimize detection and enumeration of tetramer-positive events, our analysis protocol includes multiple levels of inclusion and exclusion gating strategies to minimize background. As shown, side scatter area versus side scatter width plot is used to exclude cell aggregates. The remaining single-cell events are analyzed on a plot of non–T-cell lineages (all labeled by Pacific blue), and dead cells were stained by 4,6 diamidino-2-phenylindole (dump channel) vs CD3, allowing for T-cell events to be identified. CD8⁺ T cells specific for influenza A–Matrix1_58-66 tetramer within the enriched fraction are indicated, with minimal background staining in the CD8⁻ CD3⁺ T-cell population. (C) PBMCs (2 × 10⁵) from pre-enriched and depleted fractions from influenza A–Matrix1_58-66 (flu) or MART1_26-35 TAME were plated for IFNγ ELISPOT analysis. Cells were stimulated by indicated peptides. Data from triplicate wells were averaged, and errors bars indicate SEM. Data shown are representative of 2 independent experiments.