75 × 107cells per ml. A volume of 0.2 ml (3.5 × 106cells) tumor cell suspension was injected subcutaneously ventral to the right axilla of the mice (C57BL/6 for EL4, Kunming mice for S180). Mice were monitored for tumor burden by measuring the tumor size daily using a vernier calliper. Irradiation began when the tumor diameter attained 1.0 cm. Preparation of99mTc-HYNIC-Annexin V Human annexin V freeze-dried powder was purchased from Beijing Huada Protein Development Center Co. Ltd (Beijing, China). Human annexin V was conjugated with hydrazinonicotinamide (HYNIC), using methods described by Blankenberg et al. . Derivatized HYNIC-annexin V was radio-labelled with a99mTc tricine precursor
complex according to literature methods see more [5,
9–11]. Ispinesib purchase After chelating with the99mTc tricine precursor complex, the radio-labeling efficiency was measured by using thin-layer chromatography Silica Gel (TLC-SG), with methyl ethyl ketone and normal saline as the developing solvent. The radiochemical purity of the tracer product was then measured with High Performance Liquid Chromatography. The radio-labelled material, prepared as described above, was diluted to have specific activities ranging from 400-800 MBq μg-1 1 ml-1 which was ready for use. Tumor irradiation The tumor-bearing mice were randomly divided into an imaging group which was irradiated and imaged using99mTc-HYNIC-Annexin V, and an selleck inhibitor observation group which was only observed for tumor regression after single-dose irradiation. The EL4 lymphoma imaging group was subdivided into 4 single-dose levels: 0, 2, 4, and 8 Gy, while the S180 sarcoma imaging group received only 2 dose levels (0 and 8 Gy),
with 4 mice each level. The observation only groups of EL4 lymphoma and S180 sarcoma both received the same dose levels of 0 Gy or 8 Gy (4 mice each level). The tumors were irradiated with the 4 ADAMTS5 MV X-rays (SSD 100 cm, 1.5 cm × 1.5 cm portal) with a 0.5 cm thick tissue-equivalent material applied to the tumor surface. The mice were anesthetized before irradiation by intraperitoneal injection of 0.15 ml of 0.7% pentobarbital and immobilized with tapes. Experiments were repeated three times. 99mTc-HYNIC-annexin V imaging of radiation-induced apoptosis At 24 hours after radiation, 0.2 ml (4-8 MBq) of the prepared99mTc-HYNIC-annexinV was injected into each mouse in the imaging groups through the tail vein. Planar images were obtained 2 hours later, using a single-head γ camera (Meridian Philips Medical Systems) equipped with a parallel-hole collimator. The energy window was centered at 140 keV with a window width of 20%, and the matrix was to 256 × 256 with a magnification factor of 3.0. The acquisition time was 1 min/image. The tumor size of mice in the observation groups was measured daily after irradiation.