Cyan flow cytometry

In order to identify human EPCs, 2 × 10⁵ cells were suspended in 50 μL fluorescence-activated cell sorting (FACS) buffer (PBS containing 0.5% FBS) then stained with 0.2 mg/mL of each of the following anti-human antibodies: CD31-PE (LifeSpanBioSciences, Seattle, WA, USA), CD34-BV421 (BD Biosciences, San Jose, CA, USA), CD45-BB515 (BD Biosciences), VEGFR-2 PE (R&D Systems, Minneapolis, MN, USA), CD14-FITC (BD Biosciences), and CXCR4-PE (BioLegend, San Diego, CA, USA). OneComp eBeads (Carlsbad, CA, USA) were first stained with 1 μL of each different fluorochrome then used as single-color compensation controls. An unstained sample served as a reference for positive staining. Cells were analyzed using a Cyan flow cytometry (Beckman Coulter, Brea, CA, USA). Single cell data was analyzed using FlowJo, LLC software that gave a percentage for positive cells.

The binding of mouse PE-conjugated DR4 and DR5 specific antibodies was utilized to quantitate the surface expression of DR4 and DR5 according to manufacturer’s guidelines (R&D systems, Minneapolis, MN, USA). Briefly, 1 × 10⁶ DU145 cells were harvested by treatment with PBS-0.5 mM EDTA for 10 min at room temperature, and then washed twice with flow cytometry staining buffer (FCSB). Cells were resuspended in FCSB, incubated with FcR blocking reagent for 15 min at room temperature followed by incubation with PE-conjugated DR4 or DR5 specific antibodies or PE-conjugated isotype control IgG for 45 min at 4 °C. Cells were washed twice with FCSB and examined by CyAn flow cytometry (Beckman Coulter, Brea, CA, USA).