Control rat igg antibody

ACT in B6 nude mice, in vivo vaccination, PLX4720 treatment, anti-VEGF blocking antibody treatment, and in vivo Bioluminescence Imaging were performed as previously described (5 (link), 27 (link)). GSK2636771 and BKM120 (Chemie Tek) were suspended in 1% (w/v) methylcellulose and administered to mice daily by oral gavage at a dose of 30mg/kg and 60mg/kg respectively. For the spontaneous tumor model, Tyr:CreER; PTEN^lox/lox; BRAF ^V600E/+ mice on a C57BL/6 background (6–8 weeks of age) were treated with 4-hydroxytamoxifen to induce the expression of Cre as previously described (11 (link)). The anti-mouse PD-1 antibody (29F.1A12, Biolegend) was intraperitoneally injected on days 0, 2 and 4 at a dose of 100 μg/per mouse. The relevant solvent and control rat IgG antibody (Sigma) were administered to control animals.

Murine B16 melanoma cells stably expressing ovalbumin were cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin–streptomycin. To prepare cell supernatants for experiments, 5×10⁶ tumor cells were plated in 10ml medium for 24 hours before supernatants were collected. To induce murine melanoma, mice were implanted s.c. with 0.5–1×10⁶ B16 melanoma cells (0 days). Tumor growth was monitored every other day and mice were sacrificed when tumor sizes reached 20 mm in any direction, according to our IACUC-approved protocol. In some experiments, established tumors (at 7 days) were injected intratumorally (i.t.) with 2×10⁶ LPS-stimulated RV-GM-DCs, ID2-GM-DCs, Id2^+/GFP GM-DCs, Stat3-deficient GM-DCs or Stat3- and Id2-deficient GM-DCs (2×10⁶ DCs per 100 μl PBS), as indicated in the Figure legends. To distinguish immune cells of the donor versus recipient mice, CD45.1⁺ congenic animals (C57BL/6 background) were used in some experiments. In addition, some experiments included simultaneous intravenous (i.v.) injection of 10⁶ naïve (CD25⁻ CD62L⁺ CD44⁻) OT-II CD4⁺, OT-I CD8⁺ or both T cell populations, as indicated in the Figure legends. For PD-1 antibody treatments, anti-mouse PD-1 antibody (RMP1-14) (BioXCell) or control rat IgG antibody (Sigma) was injected intraperitoneally (i.p.) at the day of DC vaccination, and repeated every 3 days, for a total of 3 doses (200 μg/dose).