Biotinylated anti cd25 mab clone pc61

Splenocytes from wild-type B6 mice were depleted of CD25⁺ cells using biotinylated anti-CD25 mAb (clone PC61, eBiosciences) and streptavidin-conjugated beads (BD Biosciences). CD4⁺GFP⁺ T_reg cells were FACS-sorted from wild-type or Prkch^−/− FIG mice. 15 × 10⁶ CD25-depleted splenocytes were adoptively transferred alone or together with 0.5 × 10⁶ wild-type or Prkch^−/− CD4⁺GFP⁺ T_reg cells into recipient Rag1^−/− mice; this was done blind and assigned randomized with no pre-established inclusion/exclusion criteria. Two × 10⁵ B16-F10 melanoma cells were inoculated intradermally on the right shaved flanks the next day. Tumor size was measured using an electronic dial caliper 2-3 times/week^{14 (link)}. To achieve reasonable power, at least 5 mice/group (15 mice/experiment) were used. Additional mice were added to experiments as appropriate.