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Each sample was washed, counterstained with 0.1 ��g/ml Hoechst 33258 for 15 min, and mounted with Vectorshield mounting medium (Vector Laboratories). Fluorescence Selleckchem CP-868596 was detected using an Axiovert 200 fluorescence microscope (Carl Zeiss, Oberkochen, Germany) and quantified in Metamorph software (Molecular Devices). Quantitative analyses are expressed as a percentage of the sham-control group. The peripheral blood and bone marrow cells from the femur and tibia were obtained from normal mice that had been treated with cilostazol for 3, 7, and 14 days. Monocytes were isolated from the peripheral blood and the bone marrow by density gradient centrifugation with Histopaque-1083 (Sigma). The viable monocyte population was analyzed for the expression of Sca-1-fluorescein isothiocyanate PTPRJ (FITC; e-Bioscience, San Diego, CA) and vascular endothelial growth factor receptor-2-phycoerythrine (VEGFR-2 or Flk-1; e-Bioscience). Isotype-identical antibodies (e-Bioscience) served as control. Single- and two-color flow cytometric analyses were performed by a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) quantifying 1 �� 104 events using gates to exclude nonvisible cells. Data were evaluated in Cellquest software (ver. 3.3; Becton Dickinson). Cilostazol was donated by Otsuka Pharmaceutical Co. Ltd. (Tokushima, Japan) and was dissolved in DMSO. Data are expressed as mean �� SEM. The changes in variable parameters between drug-treated www.selleckchem.com/products/Lapatinib-Ditosylate.html and vehicle groups were analyzed by the unpaired Student's t-test. Analysis of each individual comparison (3, 7, 14, and 28 days) was performed by one-way ANOVA followed by Tukey's multiple-comparisons tests. Statistical analysis was performed in Sigmastat software (Systat Software, Point Richmond, CA). A value of P