Anti cd8 conjugated magnetic beads

iCD103 DCs collected on day 13 or 14 of culture were treated with indicated amount of poly (I:C) or IL17A for 24 hours, washed, and placed in coculture assays with splenic CD8⁺ T cells (at 1:10 ratio). CD8⁺ T cells were isolated using anti-CD8 conjugated magnetic beads (Miltenyi Biotec) and were isolated with an autoMACS cell separator. In some experiments when OT-I cells were used, iCD103 DCs were pretreated with ovalbumin (10 μg/ml) for 3 hours and washed three times with PBS before coculture with OT-I T cells. ELISA (BD BioSciences) was used for the measurement of concentration of IFNγ in the supernatant collected from cultured cells.

Cell suspensions were prepared from spleen, liver, thymus, blood and peripheral lymph nodes as described previously ^{47 (link)}. CD8⁺ T cells were purified using anti-CD8 conjugated magnetic beads (Miltenyi, France). Enriched CD8⁺ T cells fraction was then stained for NK1.1⁺ and CD8⁺ as well as CD44, CD62L or LCMV specific tetramers or Thy1.2 when indicated and sorted using a two laser BD FACS Aria (BD Biosciences) cell sorter/analyzer. 500–1000 of purified naïve OT-1 cells were transferred i.v. into mice.