Complete freund s adjuvant incomplete freund s adjuvant

Animal care and use were in accordance with institutional guidelines. Female FVB/N-TgN (MMTVneu)-202Mul mice (Tg-MMTVneu) (6-8 weeks old; mean weight: 18.5 g, range: 15.4-23.1 g) (Jackson Laboratory) were immunized with IGFBP-2 DNA constructs or pUMVC3 vector alone (50 μg plasmid) as a mixture in complete Freund's adjuvant/incomplete Freund's adjuvant (Sigma). Three immunizations were given two weeks apart. For tumor challenge, a syngeneic mouse mammary tumor cell line, MMC, (0.5 x 10⁶ cells) was implanted into the mammary fat pad two weeks after the last vaccine or seven days before T-cell adoptive transfer (n=10/group) (20 (link)). Tumors were measured as previously described (16 (link)). Briefly, measurements on length, width, and depth were assessed by Vernier caliper 2-3x per week by the same technician at each time point. The measurements were multiplied by the volume constant of an ellipsoid shape, or π/6, in order to calculate tumor volumes. All tumor growth is presented as mean tumor volume (mm³ ± SEM). Data are representative of two independent experiments.
For adoptive transfer, 5x10⁶ IGFBP-2 N-terminal- or C-terminal-specific T-cells were transferred into tumor-bearing mice by i.v. tail vein injection. The same number of splenocytes derived from naïve mice were used as a control infusion. Data are representative of two independent experiments.

Mice were immunized subcutaneously using a 26 ½ G needle. Each mouse was injected with 50 µl of a HIF-1α peptide pool (p38-53, p60-82, and p93-117; 50 μg each) as a mixture in complete Freund’s adjuvant/incomplete Freund’s adjuvant (Sigma). To generate an effective immune response to an overexpressed self-antigen, three immunizations were given two weeks apart (14 (link)). For tumor challenge, the corresponding syngeneic mouse mammary tumor cell line (0.5 × 10⁶ cells) was implanted into the mammary fat pad two weeks after the last vaccine (n=10/group) (14 (link)). Tumors were measured as previously described (13 (link)). All tumor growth is presented as mean tumor volume (mm³ ± SEM). Data are representative of three independent experiments.
At study termination, brain, kidney, liver, lung and heart were collected in formalin. Sections were stained with hematoxylin and eosin and toxicities were determined by a certified veterinary pathologist. Heparinized and non-heparinized blood was analyzed for complete blood count and serum chemistries, respectively, by Phoenix Central Laboratories, Everett, WA.