Pe anti human cd184 cxcr4 antibody

For analysis of a cancer stem cell phenotype (CD133+/CD44+), overexpression of CXCL12γ in PCa or control cells (PC3, DU145) (1 × 10⁵) were seeded onto 12-well culture plates and were cultured for 4 days. The cells were incubated with PE-anti-CD133 antibody (cat. 130–080-901, Miltenyi Biotec, San Diego, CA) and APC-anti-CD44 antibody (cat. 559942, BD Biosciences, San Jose, CA) for 20 min at 4°C. For CXCR4 positive cell analysis, the cells were incubated with PE-anti-human CD184 (CXCR4) antibody (cat. 306506, BioLegend, San Diego, CA) or mouse IgG-PE (cat. 130–092-212, Miltenyi Biotec) for 20 min at 4°C. The CD133+/CD44+ or CXCR4 positive fractions were analyzed with a FACS Aria High-Speed Cell Sorter (BD Biosciences). Apoptosis was measured by flow cytometry (FACSAria dual laser flow-cytometer, Becton Dickinson, Mountainview, CA) using PE Annexin V Apoptosis Detection Kit I (cat. 559763, BD Biosciences, San Jose, CA). The PCa cells were pretreated AMD3100 (5μg/ml) or siCXCR4 and treated with of docetaxel (Taxotere; 0.5–1μg/ml, Hospira, Lake Forest, IL). In some cases, the PCa cells were treated with XTANDI® (enzalutamide; 0.5μg/ml)‎ (Selleck Chemicals, Houston, TX).