Identification of Cancer Stem Cells

Side population (SP) of putative CSCs were performed as previously described [18 (link), 19 (link)]. Briefly, 1 × 10⁶ cells/ml of human PC or syngeneic-derived cancer cells were washed with PBS, suspended in DMEM containing 10% FBS, and incubated with 2 μg/ ml of Hoescht 33342 (AnaSpec Inc., Fremont, CA, USA) fluorescent dye for 90 minutes at 37°C in the dark with intermittent shaking for 90 minutes at 37°C. Cells were incubated with 100 μM of Verapamil (Sigma, St Louis, MO, USA) for 20 minutes at 37°C with intermittent shaking prior to Hoescht 33342 staining. After incubation, cells were immediately washed thrice with cold PBS and resuspended for counterstaining with 1 mL of culture media containing 10 μg/ml of propidium iodide to label dead cells. After gating, SP and non-SP cells were analyzed using BD LSR II Green flow cytometer (BD Biosciences, San Jose, CA).