Balb canncrlcrlj mice

To produce new anti-insoluble fibrin mAbs, 6-week-old BALB/cAnNCrlCrlj mice (Charles River Japan, Yokohama, Japan) or rats were immunized intraperitoneally with an emulsion of Freund's complete adjuvant (DIFCO, New Jersey, USA) and a saline solution containing 50 μg of the epitope peptide from the fibrinogen β chain that we previously discovered [7 (link)]. Three to five successive booster injections were administered intraperitoneally at 2-week intervals using the same amount of antigen in an adjuvant system (Merck, Hessen, Germany). A final boost was provided by administering the same amount of antigen intravenously. Animals were sacrificed under anesthesia, and the iliac lymph node from rats or spleen from mice was extracted and cells were fused to the P3X63-Ag8.653 myeloma cell line. Then, primary hybridoma cells were selected with HAT medium and cloned by limited dilution. Hybridoma cells were acclimated to appropriate serum-free media for large scale culture. Supernatant titer was confirmed using the Human IgG ELISA Kit (Bethyl Laboratories, Massachusetts, USA) and mAbs were obtained by affinity purification and size exclusion chromatography. The isotypes of the mAbs were determined using IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche, Basel-Stadt, Switzerland).

Nude mice (CAnN.Cg-Foxn1nu/CrlCrlj; female, 5–6 weeks old) were obtained from Charles River Laboratories Yokohama, Japan. Cancer cells (5 × 10⁶ cells/head) were implanted s.c. When the tumor volume reached 100–300 mm³, mice were randomized into treatment groups. Lenvatinib or golvatinib were given orally once a day. The tumor volume was calculated by using the following formula: tumor volume (mm³) = 1/2 × length (mm) × width (mm)². For the isolation of splenic macrophages, BALB/cAnNCrlCrlj mice (female, 5–6 weeks old) were obtained from Charles River Laboratories. All of the animal experiments were carried out in accordance with the guidelines for animal experiments of Eisai Co., Ltd.