However, the detailed anti-apoptotic signaling mechanisms of maxadilan are not fully elucidated

included in groups 6-8. Group 1 was the nave control group; group 2 received a hind limb ischemia operation at the beginning of week 1 of the experiment; group 3 received a hind limb ischemia operation at the begin of week 1 and tail vein injection of 1 g/kg body weight of GroEL1 once a week throughout the experiment; group 4 received a hind limb ischemia operation at the beginning of week 1 and tail vein injection of 2 g/kg BW of GroEL1; group 5 received a hind limb ischemia operation at the beginning of week 1 and tail vein injection of 4 g/kg BW of GroEL1; group 6 was the nave control group; group 7 received a hind limb ischemia operation at the beginning of week 1 of the experiment; group 8 received a hind limb ischemia operation at the beginning of week 1 and tail vein injection of 4 g/kg body weight of GroEL1. Mouse Ischemic Hind Limb Model At the beginning of the experiments, unilateral hind limb ischemia was 2181489 induced in six-week-old mice by ligating and excising the right femoral artery as previously described. Briefly, the animals were anesthetized by intraperitoneal injection of Xylocaine plus Zoletil. The proximal and distal portions of the femoral artery were ligated by silk thread, and we cut the blood vessel approximately 0.2 cm. Hind limb blood perfusion was measured with a laser Doppler perfusion imager system before and after the surgery and was then followed up once every two weeks. The animals were sacrificed by cervical dislocation without sedation at the end of the eight experimental weeks. To avoid the influence of ambient light and temperature, the results are expressed as the ratio of perfusion in the right versus left limb. Materials and Methods Flow Cytometry Animals and GroEL1 Administration All male C57BL/6 mice were purchased from BioLASCO Taiwan Co., Ltd. The male C57BL/10ScNJ mice were purchased from the Jackson Laboratory. All animals were treated according to protocols approved by the Institutional Animal Care Committee of the Taipei Medical University. The experimental procedures and animal care conformed to the “Guide for the Care and Use of Laboratory Animals” published To investigate the mobilization of EPCs, 2578618 a fluorescenceactivated cell sorting Caliber flow cytometer was used. Total 181223-80-3 custom synthesis leukocyte was isolated from a volume of 100-200 L peripheral blood by incubating with 200-400 L RBC lysis buffer, and washing twice in phosphate-buffered saline. Then total leukocyte was incubated with fluorescein isothiocyanate conjugated anti-mouse CD34, allophycocyanin -conjugated anti-mouse Flk-1, and phycoerythrin -conjugated anti-mouse Sca-1 antibodies 2 C. pneumoniae Impairs EPCs Function . Isotype-identical antibodies served as controls. Each analysis included 150,000~300,000 total leukocyte. Circulating EPCs were considered to be from the monocytes population and were gated with triple positivity for CD34, Sca-1, and Flk-1. EPC Isolation and HCAEC Cultivation Total mononuclear cells were isolated from 40 ml of peripheral blood from healthy young male volunteers by density-gradient centrifugation with Histopaq-1077. The Institutional Review Board approved this study, and all volunteers gave written informed consent prior to all procedures. MNCs were plated in 2 ml of endothelial growth medium with supplements on fibronectin-coated six-well plates at 37C in a 5% CO2 incubator. The cultures were observed daily, and after 4 days of culture, the media were changed and nonadherent cells were removed; attached early EPC