Anti pe or anti apc magnetic beads

The frequency of Phl p 1- and Phl p 5a-specific T-cells was measured as previously described [12 (link)]. Briefly, 30 million PBMC in 200 μL T-cell culture medium were stained with 20μg/mL PE-labeled tetramers and/or APC-labeled tetramers (for ex vivo dual tetramer staining) for 100 minutes. Cells were then washed and incubated with anti-PE or anti-APC magnetic beads (Miltenyi Biotec) for 20 minutes at 4°C and a 1/100 fraction was saved for analysis; the other fraction was passed through a magnetic column (Miltenyi Biotec, Bergisch Gladbach, Germany). Bound PE- and/or APC-labeled cells were flushed and collected. Cells in the bound and precolumn fractions were stained with a panel of antibodies of interest for 20 minutes at room temperature. After staining, cells were stained with Via-probe⁺ (BD Biosciences) for 10 minutes at 4°C before flow-cytometry. Data were analyzed utilizing FlowJo (Tree Star, Ashland, Ore) gating on forward scatter/side scatter and excluding CD14⁺, CD19⁺ and Viaprobe populations. Frequency was calculated as previously described [12 (link)]. For phenotyping studies, antibodies were used against markers of interest; CCR4 (R&D systems), CD45RA (eBioscience) and CD38 (eBioscience).

Enrichment of antigen-specific CD8 T cell precursors or LCMV-specific CD8 T cells at memory time points was performed as previously described(16 (link)). Spleens were homogenized and splenocytes were labeled with PE- or APC-conjugated tetramers, followed by magnetic enrichment over columns using anti-PE or anti-APC magnetic beads (Miltenyi). Enriched samples were stained with surface antibodies, and AccuCheck counting beads (Invitrogen) were used for calculating cell number.