Anti cd90.2 pe

Three days after intraperitoneal inoculation of B6 with 2×10⁷ IU of gB/OVA-RepliVAX WN, splenocytes were harvested and DC were enriched by depletion of B, T and NK cells using anti-CD19, -CD90.2, -CD49 (DX5) microbeads (Miltenyi Biotec, Auburn, CA), following the manufacturer’s protocol. Enriched cells were then surface-stained with fluorochrome-conjugated mAb anti-CD8α (APC), -CD11c (PE) and -CD11b (PE-Cy7) (BD Biosciences) and sorted into CD11b⁺ CD11c⁺ and CD8α⁺ CD11c⁺ subpopulations using a BD FACS Aria. Serial 2-fold dilutions of CD11c⁺CD11b⁺ or CD11c⁺CD8α⁺ DC subpopulations were co-cultured with 10⁵ naïve OT-I T cells selected by using the CD8⁺ T cell Isolation Kit II (Miltenyi Biotec) and labeled with 2μM of carboxyfluorescein succinimidyl ester (CFSE) (Molecular Probes, Invitrogen) in 96-well plates. After 72h, cells were surface-stained with fluorochrome-conjugated mAb anti-CD90.2 (PE) (BD biosciences), data were acquired on a BD LSRII Fortessa, and the proliferation of T cells measured as dilution of intracellular CFSE was determined by using FlowJo software (Tree Star).