Facsaria dual laser flow cytometer

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).

BMSC^Anxa2+/+ or BMSC^Anxa2−/− (1 × 10⁵ cells/well) were seeded onto 12-well culture plates for 24 hours. GFP expressing PCa cells (1 × 10⁵ cells/well) were added to the wells and cultured together with the BMSCs for 48 hours prior to the addition of the anticancer drug, taxotere (1μg/ml, cat. NDC0409-0201-10, Hospira, Lake Forest, IL) for an additional 48 hours. Additionally, HBMECs (1 × 10⁵ cells/well) were seeded onto 12-well culture plates for 24 hours and the cells were treated with ANXA2 (1μg/ml, cat. 11-511-248-344, Genway Biotech, San Diego, CA) or CXCL12 (200ng/ml, cat. 350-NS, R&D System) for 48 hours. Apoptosis in these cultures 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). Assays were performed in triplicate and the results are representative of three independent experiments. In tissue sections from mice inoculated with human PCa, apoptosis of PCa cells and endothelial cells in the tumors was measured by TUNEL staining (cat. 11684817910, Roche, Branford, CT).